The accumulation of undigestible autofluorescent material (UAM), termed lipofuscin in vivo, is a hallmark of aged RPE. Lipofuscin derives, in part, from the incomplete degradation of phagocytized photoreceptor outer segments (OS). Whether this accumulated waste is toxic is unclear. We therefore investigated the effects of UAM in highly differentiated human fetal RPE (hfRPE) cultures. METHODS. Unmodified and photo-oxidized OS were fed daily to confluent cultures of ARPE-19 RPE or hfRPE. The emission spectrum, composition, and morphology of resulting UAM were measured and compared to in vivo lipofuscin. Effects of UAM on multiple RPE phenotypes were assessed. RESULTS. Compared to ARPE-19, hfRPE were markedly less susceptible to UAM buildup. Accumulated UAM in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. UAM accumulation mildly reduced transepithelial electrical resistance, ketogenesis, certain RPE differentiation markers, and phagocytosis efficiency, while inducing senescence and rare, focal pockets of epithelial-mesenchymal transition. However, it had no effects on mitochondrial oxygen consumption rate, certain other RPE differentiation markers, secretion of drusen components or polarity markers, nor cell death. CONCLUSIONS. hfRPE demonstrates a remarkable resistance to UAM accumulation, suggesting mechanisms for efficient OS processing that may be lost in other RPE culture models. Furthermore, while UAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may adequately adapt to and tolerate lipofuscin accumulation.

have patents filed for the use of suPAR levels in the management of cardiovascular disease and the use anti-suPAR therapies as a strategy to prevent and treat atherosclerosis. SSH and JR are scientific advisory board members of Walden Biosciences, a company devising therapeutics targeting suPAR in kidney disease. GH, MEH, JBN and LAL receive salary, stocks and stock options from Regeneron Pharmaceuticals, Inc. JEO is a co-founder, shareholder, and chief scientific officer of Virogates and a named inventor on patents related to suPAR.

The daily shedding and renewal of photoreceptor outer segments (OS) is critical for maintaining vision. This process relies on the efficient uptake, degradation, and sorting of shed OS material by the retinal pigment epithelium (RPE). Poor OS degradation has been linked to retinal degenerations such as Stargardt disease and may contribute to macular degeneration. While primary human fetal RPE cultures have emerged as a valuable model of in vivo human RPE function, surprisingly few studies have utilized the model for tracking the degradation and fate of OS components in the RPE. Here, we establish an improved platform for studying this topic by modifying existing protocols and creating new methods. Our human fetal culture model facilitates studies of RPE secretion in response to OS ingestion, preserves RPE differentiation and polarization during live-cell imaging of OS phagocytosis, and minimizes costs. We optimize Mer tyrosine kinase-dependent OS phagocytosis assays specifically in human fetal cultures and provide a simple and accurate method for measuring total OS consumption by the RPE. Finally, we utilize chemical transfection, dextran labeling, and immunocytochemistry to evaluate key players in OS degradation, including lysosomes and autophagy proteins. To facilitate quantification of autophagy vesicles, we develop customized image analysis macros in the Fiji/ImageJ software environment. These protocols will facilitate a broad range of studies in human fetal RPE cultures aimed at determining the ultimate fate of OS components after ingestion, a critical step in understanding the pathogenesis of numerous retinal diseases.