Testing Mitochondrial-Targeted Drugs in iPSC-RPE from Patients with Age-Related Macular Degeneration

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. No universally effective treatments exist for atrophic or "dry" AMD, which results from loss of the retinal pigment epithelium (RPE) and photoreceptors and accounts for ≈80% of all AMD patients. Prior studies provide evidence for the involvement of mitochondrial dysfunction in AMD pathology. This study used induced pluripotent stem cell (iPSC) RPE derived from five AMD patients to test the efficacy of three drugs (AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), Metformin, trehalose) that target key processes in maintaining optimal mitochondrial function. The patient iPSC-RPE lines were used in a proof-of-concept drug screen, utilizing an analysis of RPE mitochondrial function following acute and extended drug exposure. Results show considerable variability in drug response across patient cell lines, supporting the need for a personalized medicine approach for treating AMD. Furthermore, our results demonstrate the feasibility of using iPSC-RPE from AMD patients to develop a personalized drug treatment regime and provide a roadmap for the future clinical management of AMD.

Keywords: age-related macular degeneration; human-induced pluripotent stem cells; personalized drug testing; retinal pigment epithelium.

Figure 1

Pathway to precision medicine for patients with age-related macular degeneration. Conjunctival biopsies from patients in the AMD clinic provide somatic cells for patient-specific iPSC- line derivation and differentiation of iPSC-RPE for testing drugs to restore or protect mitochondrial function and provide clinical benefit. Dark gray arrows indicate steps included in the current study. Light gray arrows represent future steps in personalized treatment.

Figure 2

iPSC-RPE derived after reprogramming conjunctival cells obtained from patients with AMD. (A) Phase microscopy image showing that confluent iPSC-RPE lines form a monolayer with cobblestone appearance and have pigmentation. Scale bar = 100 µm. (B) Confocal microscopy image of iPSC-RPE cultured on a transwell insert. En face views of the RPE monolayer shown as maximum intensity projection through the z-axis. Bestrophin (red) is expressed on the basal surface. ZO-1 (green) marks cell borders. Nuclei are stained with DAPI (blue). Scale bar = 40 µm. (C) Results from ELISA analysis of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A) content measured in apical (top) and basal (bottom) media from iPSC-RPE (n = 5) grown on transwells. Mean ± SEM. (D) iPSC-RPE cultures express prototypic RPE proteins as demonstrated on Western immunoblots. Molecular mass for each protein is shown on the left. HR is a homogenate of RPE tissue from a human donor. Stain-free image is loading control. (E) Representative data from FACS analysis measuring the phagocytosis of FITC-labeled OS by RPE. Dot plots and histograms for cells without and with the addition of OS are shown.

Figure 3

Testing mitochondrial function in iPSC-RPE from patients with AMD. (A) Example trace associated with Cell Mito Stress Test (CMST). Analysis of oxygen consumption rate (OCR) following injections of oligomycin (oligo), FCCP, and antimycin A + rotenone (AA + Rot) to perturb mitochondrial function. Calculation of the basal respiration (BR), maximal respiration (MR), spare respiratory capacity (SRC), and ATP-linked respiration (ATP) is shown. (B) Traces from CMST of OCR for patient-specific iPSC-RPE (five patients, seven lines). (C) Parameters of mitochondrial function were calculated from data shown in (B). Mean ± SEM. Numbers in brackets indicate coefficient of variation (CV) of OCR for two cell lines from the same patient.

Figure 4

Characterization of drug treatment effect using AMD donor iPSC-RPE cells. (A) Schematic of AMPK activation and regulation of metabolism. (B) iPSC-RPE cells (n = 4 donors) were treated with compounds (AICAR, Metformin, or trehalose) for 24 or 48 h. Mitochondrial function was evaluated using an XFe96 Extracellular Flux Analyzer. Calculated fold change values of mt function (compared to untreated controls) for the four lines are shown. BR = basal respiration; MR = maximal respiration; SRC = spare respiratory capacity; ATP = ATP-linked respiration. (C,D) Protein content in lysates from iPSC-RPE cells (n = 4 donors) following incubation with 500 µM AICAR (C) or 2 mM Metformin (D). Calculated fold change values are shown (no treatment = 1, dashed line). (E) ECAR was measured during CMST assays (n = 4 donors). (F) ATP Rate Assay was performed on iPSC-RPE (n = 4) after Metformin treatment for 48 h. (G) iPSC-RPE (n = 4 donors) were treated with 100 mM trehalose for 48 h. Content of autophagy-related proteins was determined in treated cells relative to untreated cells (dashed line). LC3-II/I, Lysosomal-Associated Membrane protein 1 (LAMP1), Cathepsin D (Cath D). (H) Maximal projection of z-stack images of LysoTracker™ staining (red) labeling lysosomes in untreated (NT) and trehalose treated (Treh) iPSC-RPE cells. Nuclei are stained with DAPI (blue). Scale bar = 100 µm. Data in (A–G) are mean ± SEM. One-sample t-tests were used to compare treatment to no treatment in (C,D,G). Unpaired t-tests were used to compare treatment to NT in (A,E,F). ** p < 0.01, * p < 0.01, # p < 0.1.

Figure 5

Testing AMPK activators using patient-specific iPSC-RPE. In two separate experiments, iPSC-RPE cells from patients with AMD (n = 5) were treated with (A) AICAR or (B) Metformin for 48 h or 3 times per week for 3 weeks. Following treatment, parameters of mt function were calculated from OCR measured using an XFe96 Extracellular Flux Analyzer. OCR data were normalized to cell count per well. Graphs show fold change relative to each donor’s no-treatment control (dashed line). Blue data points indicate response from cells that exhibited improved mt function. Red data points indicate response from cells that exhibited decreased mt function. BR = basal respiration; MR = maximal respiration; SRC = spare respiratory capacity; ATP = ATP-linked respiration. * p < 0.05, ** p < 0.01, # p < 0.1 determined by unpaired t-tests of raw OCR values (average no treat OCR vs. average treatment OCR).

Figure 6

Testing an autophagy inducer using AMD patient-specific iPSC-RPE. iPSC-RPE cells from patients with AMD (n = 4) were treated with trehalose for 48 h. Following treatment, parameters of mt function were calculated from OCR measured using an XFe96 Extracellular Flux Analyzer. OCR data were normalized to cell count per well. Graphs show the fold change relative to each donor’s no-treatment control (dashed line). Blue data points indicate response from cells that exhibited improved mt function. Red data points indicate response from cells that exhibited decreased mt function. BR = basal respiration; MR = maximal respiration; SRC = spare respiratory capacity; ATP = ATP-linked respiration. * p < 0.05, ** p < 0.01 determined by unpaired t-tests of raw OCR values (average no treat OCR vs. average treatment OCR).