Stem cell-derived retinal pigment epithelium from patients with age-related macular degeneration exhibit reduced metabolism and matrix interactions

Abstract

Modeling age‐related macular degeneration (AMD) is challenging, because it is a multifactorial disease. To focus on interactions between the retinal pigment epithelium (RPE) and Bruch's membrane, we generated RPE from AMD patients and used an altered extracellular matrix (ECM) that models aged Bruch's membrane. Induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from AMD patients or age‐matched (normal) controls. RPE derived from iPSCs were analyzed by morphology, marker expression, transepithelial electrical resistance (TER), and phagocytosis of rod photoreceptor outer segments. Cell attachment and viability was tested on nitrite‐modified ECM, a typical modification of aged Bruch's membrane. DNA microarrays with hierarchical clustering and analysis of mitochondrial function were used to elucidate possible mechanisms for the observed phenotypes. Differentiated RPE displayed cell‐specific morphology and markers. The TER and phagocytic capacity were similar among iPSC‐derived RPE cultures. However, distinct clusters were found for the transcriptomes of AMD and control iPSC‐derived RPE. AMD‐derived iPSC‐RPE downregulated genes responsible for metabolic‐related pathways and cell attachment. AMD‐derived iPSC‐RPE exhibited reduced mitochondrial respiration and ability to attach and survive on nitrite‐modified ECM. Cells that did attach induced the expression of complement genes. Despite reprogramming, iPSC derived from AMD patients yielded RPE with a transcriptome that is distinct from that of age‐matched controls. When challenged with an AMD‐like modification of Bruch's membrane, AMD‐derived iPSC‐RPE activated the complement immune system.

Altered Protein Function Caused by AMD-associated Variant rs704 Links Vitronectin to Disease Pathology

“…Searching for in vitro model systems applicable to analyzing an influence of rs704 on vitronectin functionality, we tested endogenous vitronectin expression in different retinal and non-retinal cells frequently used as model systems to investigate AMD-associated molecular pathomechanisms or functionality of retinal disease-related proteins. 12 , 34 , 44 These included human RPE cell lines ARPE-19 and hiPSC–RPE, human retinoblastoma cell lines Y79 and WERI-Rb1, endothelial cell line HUVEC, and HEK293 cells.…”

“…ECM produced by ARPE-19 cells is composed of characteristic Bruch's membrane proteins 38 , 39 and supports primary RPE cells to acquire and maintain the RPE phenotype. 38 Therefore, despite the limitation of ARPE-19 cells for studying certain aspects of RPE function due to low polarization, pigmentation, and transepithelial resistance, 71 – 73 they frequently serve as a model system to recreate Bruch's membrane pathology in culture 12 , 38 , 39 , 74 , 75 (reviewed in Fields et al 76 ). In our study, heterologous expression of vitronectin by ARPE-19 cells increased deposition and clustering of ECM constituents such as fibronectin, elastin, and collagen VI.…”

“…[8][9][10] In addition, the cytoplasm of drusen-associated retinal pigment epithelial (RPE) cells from human donors was found to contain elevated amounts of vitronectin. 11 In vitro studies have shown an upregulation of vitronectin mRNA expression in human induced pluripotent stem cells (hiPSCs)-RPE cells derived from AMD patients, 12 as well as an upregulation of the vitronectin protein in complement-stimulated, immortalized RPE (ARPE- 19) cells. 13 Vitronectin is a secreted glycoprotein circulating in blood serum but has also been found to be deposited in the ECM of various tissues.…”

Experimental Models in Neovascular Age Related Macular Degeneration

“…An accumulation of autophagosomes and a reduction in autophagic activity were also observed. Furthermore, iPSC derived RPE cells isolated from vAMD patients expressed higher levels of gene coding for the complement [ 25 ] and had more difficulty in attaching and surviving on nitrite-modified extra cellular matrix than cells from healthy donors.…”

A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium

“…The RPE forms a monolayer of highly specialized pigmented cells located between the neurosensory retina and the choroidal vasculature and plays a pivotal role in the visual cycle and maintaining the health of the outer retina [ 23 ]. Consequently, extensive research efforts have focused on the cell biology of the RPE [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ] including studies examining the effects of zinc supplementation [ 36 , 37 , 38 ]. These studies resulted in mixed outcomes [ 12 , 39 , 40 , 41 , 42 , 43 ] likely due to the availability of zinc to exert biological function being affected by differing compositions of culture media [ 44 ].…”