A mid-throughput mitochondrial membrane potential assay in iPSC-derived RPE cells optimised for compound screening

Age-related macular degeneration (AMD) is characterised by retinal pigment epithelium (RPE) dysfunction and degeneration, leading to secondary photoreceptor loss and central vision impairment. Mitochondrial dysfunction is implicated in AMD pathogenesis, making mitochondrial pathways attractive therapeutic targets. While induced pluripotent stem cell (iPSC)-derived RPE cells are relevant to modelling this condition and for drug screening, intrinsic pigmentation complicates optical readouts and limits phenotypic screening. We optimised a mid-throughput, fluorescence-based assay to quantify mitochondrial membrane potential (ΔΨm) in iPSC-derived RPE cells, using tetramethylrhodamine ethyl ester (TMRE), with conditions adapted to minimise pigment-associated signal interference and compatible with automated 96-well screening. Using a carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone(FCCP)-challenge paradigm, we screened 160 FDA-approved compounds and prioritised a small set of reproducible TMRE-preserving hits under FCCP-induced stress. Matched viability profiling indicated compound- and line-dependent cytotoxicity at some effective doses, supporting the need for orthogonal triage when interpreting TMRE rescue. Follow-up transcriptomic profiling by Multiplex Analysis of Cells sequencing (MAC-seq) revealed inter-line variability in transcriptional responses, including cases where functional ΔΨm preservation occurred with minimal or no detectable differential expression under the tested conditions. Together, this work establishes a practical FCCP-challenge phenotypic screening platform for ΔΨm in low-pigment iPSC-derived RPE cells and supports combining functional, viability, and transcriptomic readouts to prioritise candidate modulators of mitochondrial stress resilience in genetically diverse backgrounds.