iPSC-derived Retinal Pigment Epithelium Research Articles

Dual CRALBP isoforms unveiled: iPSC-derived retinal modeling and AAV2/5-RLBP1 gene transfer raise considerations for effective therapy

Inherited retinal diseases (IRDs) are characterised by progressive vision loss. There are over 270 causative IRD genes and variants within the same gene can cause clinically distinct disorders. One example is RLBP1 that encodes CRALBP. CRALBP is an essential protein in the rod and cone visual cycles that take place primarily in the retinal pigment epithelium (RPE) but also in Müller cells of the neuroretina. RLBP1 variants lead to three clinical subtypes: Bothnia dystrophy, retinitis punctata albescens and Newfoundland rod-cone dystrophy. We modelled RLBP1-IRD subtypes using patient-specific iPSC-derived RPE and identified pathophysiological markers that served as pertinent therapeutic read-outs. We developed an AAV2/5-mediated gene supplementation strategy and performed a proof-of-concept study in the human models, which was validated in vivo in an Rlbp1-/- murine model. Most importantly, we identified a previously unsuspected smaller CRALBP isoform that is naturally and differentially expressed both in the human and murine retina. This previously unidentified isoform is produced from an alternative methionine initiation site. This work provides further insights into CRALBP expression and RLBP1-associated pathophysiology and raises important considerations for successful gene supplementation therapy.

Evaluating precision medicine approaches for gene therapy in patient-specific cellular models of Bietti crystalline dystrophy.

Patient-specific induced pluripotent stem cell-derived (iPSC-derived) cell lines allow for therapies to be tailored to individual patients, increasing therapeutic precision and efficiency. Bietti crystalline dystrophy (BCD) is a rare blinding disease estimated to affect about 67,000 individuals worldwide. Here, we used iPSC-derived retinal pigment epithelium (iRPE) cells from patients with BCD to evaluate adeno-associated virus-mediated (AAV-mediated) gene augmentation therapy strategies. We found that BCD iRPE cells were vulnerable to blue light-induced oxidative stress and that cellular phenotype can be quantified using 3 robust biomarkers: reactive oxygen species (ROS), 4-hydroxy 2-nonenal (4-HNE) levels, and cell death rate. Additionally, we demonstrated that AAV-mediated gene therapy can significantly reduce light-induced cell death in BCD iRPE cells. This is the first proof-of-concept study to our knowledge to show that AAV-CYP4V2 gene therapy can be used to treat light-induced RPE damage in BCD. Furthermore, we observed significant variability in cellular phenotypes among iRPE from patients with BCD of divergent mutations, which outlined genotype-phenotype correlations in BCD patient-specific cell disease models. Our results reveal that patient-specific iRPE cells retained personalized responses to AAV-mediated gene therapy. Therefore, this approach can advance BCD therapy and set a precedent for precision medicine in other diseases, emphasizing the necessity for personalization in healthcare to accommodate individual diversity.

Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients' fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Considerations for Developing an Autologous Induced Pluripotent Stem Cell (iPSC)-Derived Retinal Pigment Epithelium (RPE) Replacement Therapy.

Cell-replacement therapies are a new class of treatments, which include induced pluripotent stem cell (iPSC)-derived tissues that aim to replace degenerated cells. iPSCs can potentially be used to generate any cell type of the body, making them a powerful tool for treating degenerative diseases. Cell replacement for retinal degenerative diseases is at the forefront of cell therapies, given the accessibility of the eye for surgical procedures and a huge unmet medical need for retinal degenerative diseases with no current treatment options. Clinical trials are ongoing in different parts of the world using stem cell-derived retinal pigment epithelium (RPE). This review focuses on scientific and regulatory considerations when developing an iPSC-derived RPE cell therapy from the development of a robust and efficient differentiation protocol to critical quality control assays for cell validation, the choice of an appropriate animal model for preclinical testing, and the regulatory aspects that dictate the final approval for proceeding to a first-in-human clinical trial.

Biallelic CLCN2 mutations cause retinal degeneration by impairing retinal pigment epithelium phagocytosis and chloride channel function

CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2and the retina is well-established in mice, as loss-of-function of CLC-2can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.

The Predicted Splicing Variant c.11+5G>A in RPE65 Leads to a Reduction in mRNA Expression in a Cell-Specific Manner.

Pathogenic variants in RPE65 lead to retinal diseases, causing a vision impairment. In this work, we investigated the pathomechanism behind the frequent RPE65 variant, c.11+5G>A. Previous in silico predictions classified this change as a splice variant. Our prediction using novel software's suggested a 124-nt exon elongation containing a premature stop codon. This elongation was validated using midigenes-based approaches. Similar results were observed in patient-derived induced pluripotent stem cells (iPSC) and photoreceptor precursor cells. However, the splicing defect in all cases was detected at low levels and thereby does not fully explain the recessive condition of the resulting disease. Long-read sequencing discarded other rearrangements or variants that could explain the diseases. Subsequently, a more relevant model was employed: iPSC-derived retinal pigment epithelium (RPE) cells. In patient-derived iPSC-RPE cells, the expression of RPE65 was strongly reduced even after inhibiting a nonsense-mediated decay, contradicting the predicted splicing defect. Additional experiments demonstrated a cell-specific gene expression reduction due to the presence of the c.11+5G>A variant. This decrease also leads to the lack of the RPE65 protein, and differences in size and pigmentation between the patient and control iPSC-RPE. Altogether, our data suggest that the c.11+5G>A variant causes a cell-specific defect in the expression of RPE65 rather than the anticipated splicing defect which was predicted in silico.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

Retinal organoids provide unique insights into molecular signatures of inherited retinal disease throughout retinogenesis.

The demand for induced pluripotent stem cells (iPSC)-derived retinal organoid and retinal pigment epithelium (RPE) models for the modelling of inherited retinopathies has increased significantly in the last decade. These models are comparable with foetal retinas up until the later stages of retinogenesis, expressing all of the key neuronal markers necessary for retinal function. These models have proven to be invaluable in the understanding of retinogenesis, particular in the context of patient-specific diseases. Inherited retinopathies are infamously described as clinically and phenotypically heterogeneous, such that developing gene/mutation-specific animal models in each instance of retinal disease is not financially or ethically feasible. Further to this, many animal models are insufficient in the study of disease pathogenesis due to anatomical differences and failure to recapitulate human disease phenotypes. In contrast, iPSC-derived retinal models provide a high throughput platform which is physiologically relevant for studying human health and disease. They also serve as a platform for drug screening, gene therapy approaches and in vitro toxicology of novel therapeutics in pre-clinical studies. One unique characteristic of stem cell-derived retinal models is the ability to mimic in vivo retinogenesis, providing unparalleled insights into the effects of pathogenic mutations in cells of the developing retina, in a highly accessible way. This review aims to give the reader an overview of iPSC-derived retinal organoids and/or RPE in the context of disease modelling of several inherited retinopathies including Retinitis Pigmentosa, Stargardt disease and Retinoblastoma. We describe the ability of each model to recapitulate in vivo disease phenotypes, validate previous findings from animal models and identify novel pathomechanisms that underpin individual IRDs.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

SummaryLeber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

Oxidative stress differentially impacts apical and basolateral secretion of angiogenic factors from human iPSC-derived retinal pigment epithelium cells

The retinal pigment epithelium (RPE) is a polarized monolayer that secretes growth factors and cytokines towards the retina apically and the choroid basolaterally. Numerous RPE secreted proteins have been linked to the pathogenesis of age-related macular degeneration (AMD). The purpose of this study was to determine the differential apical and basolateral secretome of RPE cells, and the effects of oxidative stress on directional secretion of proteins linked to AMD and angiogenesis. Tandem mass tag spectrometry was used to profile proteins in human iPSC-RPE apical and basolateral conditioned media. Changes in secretion after oxidative stress induced by H2O2 or tert-butyl hydroperoxide (tBH) were investigated by ELISA and western analysis. Out of 926 differentially secreted proteins, 890 (96%) were more apical. Oxidative stress altered the secretion of multiple factors implicated in AMD and neovascularization and promoted a pro-angiogenic microenvironment by increasing the secretion of pro-angiogenic molecules (VEGF, PTN, and CRYAB) and decreasing the secretion of anti-angiogenic molecules (PEDF and CFH). Apical secretion was impacted more than basolateral for PEDF, CRYAB and CFH, while basolateral secretion was impacted more for VEGF, which may have implications for choroidal neovascularization. This study lays a foundation for investigations of dysfunctional RPE polarized protein secretion in AMD and other chorioretinal degenerative disorders.

Modeling of mitochondrial bioenergetics and autophagy impairment in MELAS-mutant iPSC-derived retinal pigment epithelial cells

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.

Overcoming translational barriers in modeling macular degenerations.

Advances in tissue mimetics are paving the way for interrogating both the pathobiology of human disease and innovative therapeutic paradigms. In this issue of Cell Stem Cell, Manian etal. (2021) develop a novel iPSC-derived retinal pigment epithelium (RPE)-choriocapillaris (CC) complex that recapitulates key features of macular degenerations.

Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) is valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in Best Disease patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

Retinal Pigment Epithelium Regeneration by Induced Pluripotent Stem Cells; Therapeutic and Modelling Approaches on Retinal Degenerative Diseases.

Retinal degenerative diseases (RDDs) are irreversible ocular damages categorized as retinopathies. RDDs affect about 0.05% of individuals worldwide. The degenerations of RPE cells are involved in inherited and age-related RDDs. After the invention of induced pluripotent stem cells (iPSC) by Yamanaka, a promising avenue has been opened to regenerative medicine and disease modeling. Retinal pigment epithelium (RPE) degeneration related-RDDs are also affected by iPSCs. IPSC-derived RPE cells created a novel method for treating the RPE degeneration related- RDDs and retinal diseases modeling to find a new therapeutic approach or drug development. There are various studies based on iPSC-derived RPE cells reporting the investigation of the role of a specific mutation, protein, signaling pathway, etc., responsible for a type of RDD. Furthermore, iPSC-based RPE therapy is expanded to include some clinical trials. Despite the incredible growth rate in iPSC-based studies on RPE-related diseases, there are some challenges, i.e., teratoma formation potential of iPSCs, an expensive procedure of iPSC-based regeneration of RPEs, lack of a universal protocol or cellular product applicable in all patients, etc. This article reviews the iPSC-based RPE generation and their therapeutic applications, studies on RPE-related molecular and cellular pathophysiologic features of RDD in the iPSC-based models, future perspectives, and the challenges ahead.

Automating Human Induced Pluripotent Stem Cell Culture and Differentiation of iPSC-Derived Retinal Pigment Epithelium for Personalized Drug Testing.

Derivation and differentiation of human induced pluripotent stem cells (hiPSCs) provide the opportunity to generate medically important cell types from individual patients and patient populations for research and the development of potential cell therapies. This technology allows disease modeling and drug screening to be carried out using diverse population cohorts and with more relevant cell phenotypes than can be accommodated using traditional immortalized cell lines. However, technical complexities in the culture and differentiation of hiPSCs, including lack of scale and standardization and prolonged experimental timelines, limit the adoption of this technology for many large-scale studies, including personalized drug screening. The entry of reproducible end-to-end automated workflows for hiPSC culture and differentiation, demonstrated on commercially available platforms, provides enhanced accessibility of this technology for both research laboratories and commercial pharmaceutical testing. Here we have utilized TECAN Fluent automated cell culture workstations to perform hiPSC culture and differentiation in a reproducible and scalable process to generate patient-derived retinal pigment epithelial cells for downstream use, including drug testing. hiPSCs derived from multiple donors with age-related macular degeneration (AMD) were introduced into our automated workflow, and cell lines were cultured and differentiated into retinal pigment epithelium (RPE). Donor hiPSC-RPE lines were subsequently entered in an automated drug testing workflow to measure mitochondrial function after exposure to "mitoactive" compounds. This work demonstrates scalable, reproducible culture and differentiation of hiPSC lines from individuals on the TECAN Fluent platform and illustrates the potential for end-to-end automation of hiPSC-based personalized drug testing.

Transcription Factor Overexpression Drives Reliable Differentiation of Retinal Pigment Epithelium from Human Induced Pluripotent Stem Cells

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) are valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

Stem Cell Transplantation Therapy for Retinal Degenerative Diseases.

In the past decade, progress in the research on human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) has provided the solid basis to derive retinal pigment epithelium, photoreceptors, and ganglion cells from hESCs/iPSCs for transplantation therapy of retinal degenerative diseases(RDD). Recently, the iPSC-derived retinal pigment epithelium cells have achieved efficacy in treating patients with age-related macular degeneration (AMD). However, there isstill much work to be done about the differentiation of hESCs/iPSCs into clinically required retinal cells and improvement in the methods to deliver the cells into the retinaof patients. Here we will review the research advances in stem cell transplantation in animal studies andclinical trials as well as propose the challenges for improvingthe clinical efficacy and safety of hESCs/iPSCs-derived retinal neural cellsin treating retinal degenerative diseases.

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

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.