Dataset Information

IPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

ABSTRACT: Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.

SUBMITTER: Eade KT

PROVIDER: S-EPMC10145939 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

iPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Eade Kevin T KT Ansell Brendan Robert E BRE Giles Sarah S Fallon Regis R Harkins-Perry Sarah S Nagasaki Takayuki T Tzaridis Simone S Wallace Martina M Mills Elizabeth A EA Farashi Samaneh S Johnson Alec A Sauer Lydia L Hart Barbara B Diaz-Rubio M Elena ME Bahlo Melanie M Metallo Christian C Allikmets Rando R Gantner Marin L ML Bernstein Paul S PS Friedlander Martin M

The Journal of clinical investigation 20230501 9

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations an ...[more]

PMID: 37115691

Similar Datasets

Project description:BackgroundRetinitis pigmentosa (RP) is an inherited retinal disease characterized by progressive loss of photoreceptor cells. This study aim at exploring the effect of retinal pigment epithelium (RPE) derived from human-induced pluripotent stem cell (hiPSC-RPE) on the retina of retinal degeneration 10 (rd10) mice, which are characterized with progressive photoreceptor death.MethodsWe generated RPE from hiPSCs by sequential supplementation with retinal-inducing factors and RPE specification signaling factors. The three-dimensional (3D) spheroid culture method was used to obtain optimal injectable hiPSC-RPE cells. Subretinal space transplantation was conducted to deliver hiPSC-RPE cells into the retina of rd10 mice. Neurotrophic factor secretion from transplanted hiPSC-RPE cells was detected by enzyme-linked immunosorbent assay (ELISA). Immunostaining, Western blotting, electroretinography (ERG), and visual behavior testing were performed to determine the effects of hiPSC-RPE on the retinal visual function in rd10 mice.ResultsOur data demonstrated that hiPSC-RPE cells exhibited classic RPE properties and phenotype after the sequential RPE induction from hiPSCs. hiPSC-RPE cells co-cultured with mouse retinal explants or retinal ganglion cells 5 (RGC5) exhibited decreased apoptosis. The viability and functional properties of hiPSC-RPE cells were enhanced by 3D spheroid culture. Transplanted hiPSC-derived RPE cells were identified by immunostaining with human nuclear antigen staining in the retina of rd10 14 days after subretinal space injection. The pigment epithelium-derived factor level was increased significantly. The expression of CD68, microglial activation marker, reduced after transplantation. The light avoidance behavior and ERG visual function in rd10 mice improved by the transplantation of hiPSC-RPE cells.ConclusionOur findings suggest that injectable hiPSC-RPE cells after 3D spheroid culture can rescue the structure and function of photoreceptors by sub-retinal transplantation, which lay the foundation for future clinical cell therapy to treat RP and other retinal degeneration diseases.

Project description:BackgroundMitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics.MethodsWe used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p0 cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p0 cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE "cybrids." Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins.ResultsWe found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p0 and MELAS iPSC-derived RPE cells.ConclusionsOur studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype.

Project description:BackgroundAge-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. Here, we investigate how repeated wound stimulus leads to seminal changes in gene expression and the onset of a perpetual state of stimulus-independent wound response in retinal pigmented epithelial (RPE) cells, a cell-type central to the etiology of AMD.MethodsTranscriptome wide expression profiles of human fetal RPE cell cultures as a function of passage and time post-plating were determined using Agilent 44 K whole genome microarrays and RNA-Seq. Using a systems level analysis, differentially expressed genes and pathways of interest were identified and their role in the establishment of a persistent mesenchymal state was assessed using pharmacological-based experiments.ResultsUsing a human fetal RPE cell culture model that considers monolayer disruption and subconfluent culture as a proxy for wound stimulus, we show that prolonged wound stimulus leads to terminal acquisition of a mesenchymal phenotype post-confluence and altered expression of more than 40 % of the transcriptome. In contrast, at subconfluence fewer than 5 % of expressed transcripts have two-fold or greater expression differences after repeated passage. Protein-protein and pathway interaction analysis of the genes with passage-dependent expression levels in subconfluent cultures reveals a 158-node interactome comprised of two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGFβ pathway activators: TGFB1, TGFB2, INHBA, INHBB, GDF6, CTGF, and THBS1. Significantly, inhibition of TGFBR1/ACVR1B mediated signaling using receptor kinase inhibitors both forestalls and largely reverses the passage-dependent loss of epithelial potential; thus extending the effective lifespan by at least four passages. Moreover, a disproportionate number of RPE wound response genes have altered expression in neovascular and geographic AMD, including key members of the TGFβ pathway.ConclusionsIn RPE cells the switch to a persistent mesenchymal state following prolonged wound stimulus is driven by lasting activation of the TGFβ pathway. Targeted inhibition of TGFβ signaling may be an effective approach towards retarding AMD progression and producing RPE cells in quantity for research and cell-based therapies.

Project description:Oxidative cleavage of docosahexaenoate (DHA) in retinal pigmented epithelial (RPE) cells produces 4-hydroxy-7-oxohept-5-enoic acid (HOHA) esters of 2-lysophosphatidylcholine (PC). HOHA-PC spontaneously releases a membrane-permeant HOHA lactone that modifies primary amino groups of proteins and ethanolamine phospholipids to produce 2-(ω-carboxyethyl)pyrrole (CEP) derivatives. CEPs have significant pathological relevance to age-related macular degeneration (AMD) including activation of CEP-specific T-cells leading to inflammatory M1 polarization of macrophages in the retina involved in "dry AMD" and TLR2-dependent induction of angiogenesis that characterizes "wet AMD". RPE cells accumulate DHA from shed rod photoreceptor outer segments through phagocytosis and from plasma lipoproteins secreted by the liver through active uptake from the choriocapillaris. As a cell model of light-induced oxidative damage of DHA phospholipids in RPE cells, ARPE-19 cells were supplemented with DHA, with or without the lipofuscin fluorophore A2E. In this model, light exposure, in the absence of A2E, promoted the generation HOHA lactone-glutathione (GSH) adducts, depletion of intracellular GSH and a competing generation of CEPs. While DHA-rich RPE cells exhibit an inherent proclivity toward light-induced oxidative damage, photosensitization by A2E nearly doubled the amount of lipid oxidation and expanded the spectral range of photosensitivity to longer wavelengths. Exposure of ARPE-19 cells to 1 μM HOHA lactone for 24 h induced massive (50%) loss of lysosomal membrane integrity and caused loss of mitochondrial membrane potential. Using senescence-associated β-galactosidase (SA β-gal) staining that detects lysosomal β-galactosidase, we determined that exposure to HOHA lactone induces senescence in ARPE-19 cells. The present study shows that products of light-induced oxidative damage of DHA phospholipids in the absence of A2E can lead to RPE cell dysfunction. Therefore, their toxicity may be especially important in the early stages of AMD before RPE cells accumulate lipofuscin fluorophores.

Dataset Information

IPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Publications

iPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets