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Purpose: Metabolic defects in the retinal pigment epithelium (RPE) underlie many retinal degenerative diseases. This study aims to identify the nutrient requirements of healthy and diseased human RPE cells., Methods: We profiled nutrient use of various human RPE cells, including differentiated and dedifferentiated fetal RPE (fRPE), induced pluripotent stem cell-derived RPE (iPSC RPE), Sorsby fundus dystrophy (SFD) patient-derived iPSC RPE, CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and ARPE-19 cell lines using Biolog Phenotype MicroArray Assays., Results: Differentiated fRPE cells and healthy iPSC RPE cells can use 51 and 48 nutrients respectively, including sugars, intermediates from glycolysis and tricarboxylic acid (TCA) cycle, fatty acids, ketone bodies, amino acids, and dipeptides. However, when fRPE cells lose their epithelial phenotype through dedifferentiation, nutrient use becomes restricted to 17 nutrients, primarily sugar and glutamine-related amino acids. SFD RPE cells can use 37 nutrients; however, compared to cSFD RPE and healthy iPSC RPE, they are unable to use lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they show increased use of branch-chain amino acids (BCAAs) and BCAA-containing dipeptides. Dedifferentiated ARPE-19 cells grown in traditional culture media cannot use lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation toward an epithelial phenotype, restoring the ability to use these nutrients., Conclusions: Epithelial phenotype confers metabolic flexibility to healthy RPE for using various nutrients. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the potentially important roles of nutrient availability and use in RPE differentiation and diseases.

Purpose: Complement dysregulation is a key component in the pathogenesis of age-related macular degeneration (AMD) and related diseases such as early-onset macular drusen (EOMD). Although genetic variants of complement factor H (CFH) are associated with AMD risk, the impact of CFH and factor H-like protein 1 (FHL-1) expression on local complement activity in human retinal pigment epithelium (RPE) remains unclear., Methods: We identified a novel CFH variant in a family with EOMD and generated patient induced pluripotent stem cell (iPSC)-derived RPE cells. We assessed CFH and FHL-1 co-factor activity through C3b breakdown assays and measured complement activation by immunostaining for membrane attack complex (MAC) formation. Expression of CFH, FHL-1, local alternative pathway (AP) components, and regulators of complement activation (RCA) in EOMD RPE cells was determined by quantitative PCR, western blot, and immunostaining. Isogenic EOMD (cEOMD) RPE was generated using CRISPR/Cas9 gene editing., Results: The CFH variant (c.351-2A>G) resulted in loss of CFH and FHL-1 expression and significantly reduced CFH and FHL-1 protein expression (∼50%) in EOMD iPSC RPE cells. These cells exhibited increased MAC deposition upon exposure to normal human serum. Under inflammatory or oxidative stress conditions, CFH and FHL-1 expression in EOMD RPE cells paralleled that of controls, whereas RCA expression, including MAC formation inhibitors, was elevated. CRISPR/Cas9 correction restored CFH/FHL-1 expression and mitigated alternative pathway complement activity in cEOMD RPE cells., Conclusions: Identification of a novel CFH variant in patients with EOMD resulting in reduced CFH and FHL-1 and increased local complement activity in EOMD iPSC RPE supports the involvement of CFH haploinsufficiency in EOMD pathogenesis.