The retinal pigment epithelium (RPE) is a monolayer of cells underlying and supporting the neural retina. It begins as a plastic tissue, capable, in some species, of generating lens and retina, but differentiates early in development and remains normally nonproliferative throughout life. Here we show that a subpopulation of adult human RPE cells can be activated in vitro to a self-renewing cell, the retinal pigment epithelial stem cell (RPESC) that loses RPE markers, proliferates extensively, and can redifferentiate into stable cobblestone RPE monolayers. Clonal studies demonstrate that RPESCs are multipotent and in defined conditions can generate both neural and mesenchymal progeny. This plasticity may explain human pathologies in which mesenchymal fates are seen in the eye, for example in proliferative vitroretinopathy (PVR) and phthisis bulbi. This study establishes the RPESC as an accessible, human CNS-derived multipotent stem cell, useful for the study of fate choice, replacement therapy, and disease modeling.

PurposeThe goal of this study was to examine changes in the expression of transcripts and proteins associated with drusen in Age-related Macular Degeneration (AMD) after exposing human retinal pigment epithelium (hRPE) cells to chronic oxidative stress.MethodsPrimary adult human RPE cells were isolated from cadaveric donor eyes. The subpopulation of RPE stem cells (RPESCs) was activated, expanded, and then differentiated into RPE progeny. Confluent cultures of RPESC-derived hRPE and ARPE-19 cells were exposed to a regimen of tert-butylhydroperoxide (TBHP) for 1-5 days. After treatment, gene expression was measured by quantitative PCR (qPCR), protein expression was assessed by immunocytochemistry and transepithelial resistance and cell toxicity were measured.ResultshRPE cells exposed to a regimen of TBHP for 5 days upregulate expression of several molecules identified in drusen, including molecular chaperones and pro-angiogenic factors. 5-day TBHP treatment was significantly more effective than 1-day treatment at eliciting these effects. The extent of hRPE response to 5-day treatment varied significantly between individual donors, nevertheless, 6 transcripts were reliably significantly upregulated. ARPE-19 cells treated with the same 5-day stress regime did not show the same pattern of response and did not upregulate this group of transcripts.ConclusionsRPESC-derived hRPE cells change significantly when exposed to repeated oxidative stress conditions, upregulating expression of several drusen-related proteins and transcripts. This is consistent with the hypothesis that hRPE cells are competent to be a source of proteins found in drusen deposits. Our results suggest that donor-specific genetic and environmental factors influence the RPE stress response. ARPE-19 cells appear to be less representative of AMD-like changes than RPESC-derived hRPE. This adult stem cell-based system using chronic TBHP treatment of hRPE represents a novel in vitro model useful for the study of drusen formation and dry AMD pathophysiology.

To examine changes in the retinal pigment epithelium (RPE) in eyes with age-related macular degeneration (AMD) and specifically to characterize ␣B-crystallin expression in RPE cells as a biomarker in this disease. Methods: Maculae from human patients diagnosed as having AMD or from age-matched control eyes were isolated, cryosectioned, and analyzed immunohistochemically for ␣B-crystallin and for cell type-specific markers. Results: In eyes with dry and wet AMD, ␣B-crystallin was heterogeneously expressed by a subpopulation of RPE cells in the macular region (frequently in cells adjacent to drusen) and in areas of RPE hypertrophy associated with wet AMD. In contrast, ␣B-crystallin was not detected at significant levels in control RPE. Conclusion: Accompanying the formation of drusen in early-stage and late-stage AMD, RPE cells undergo change to express ␣B-crystallin. Clinical Relevance: The detection of ␣B-crystallin in the RPE of patients with early and advanced AMD implicates this as an AMD biomarker. Sporadic expression of ␣B-crystallin by RPE cells localized adjacent to drusen in early AMD indicates that changes in the gene expression of RPE cells accompany early stages of the disease and introduces novel potential targets for AMD therapy.