ABSTRACT
Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases, such as age-related macular degeneration. In-depth characterization of RPE cell product identity and critical quality attributes are needed to enhance efficacy and safety of replacement therapy strategies. Here, we characterized an adult RPE stem cell-derived (RPESC-RPE) cell product using bulk and single-cell RNA sequencing (scRNA-seq), assessing functional cell integration in vitro into a mature RPE monolayer and in vivo efficacy by vision rescue in the Royal College of Surgeons rats. scRNA-seq revealed several distinct subpopulations in the RPESC-RPE product, some with progenitor markers. We identified RPE clusters expressing genes associated with in vivo efficacy and increased cell integration capability. Gene expression analysis revealed lncRNA (TREX) as a predictive marker of in vivo efficacy. TREX knockdown decreased cell integration while overexpression increased integration in vitro and improved vision rescue in the RCS rats.
Subject(s)
Gene Expression Profiling , Neurons , Animals , Rats , Biomarkers , Epithelial Cells , Retinal PigmentsABSTRACT
How does the human eye develop in concert with the brain to create a functioning visual system? In this issue of Cell Stem Cell, Gabriel et al. (2021) report the development of eye-like structures from forebrain organoids with light sensitivity, signal processing, and connectivity, which moves us toward answering this complex question.
Subject(s)
Brain , Organoids , Humans , ProsencephalonABSTRACT
A wide variety of stem cell-derived therapies are under development for the treatment of retinal degeneration. In order to better understand patient perspectives about these therapies, we assessed risk tolerance using an in-person survey of 178 patients at an academic eye center. Risk of malignancy served as a hypothetical, readily understood, and serious adverse event to be considered in trade for potential visual improvement from a stem cell-derived treatment. The results indicate that patients were willing to trade visual improvement against a risk of malignancy that far exceeds actual risk. Two novel findings were that older patients and those with an intermediate level of visual loss were particularly risk tolerant. The quantitative survey results provide a step toward understanding patient perspectives that will, over the long term, guide the development of ocular stem cell-derived therapies.
Subject(s)
Patient Preference , Perception , Retinal Degeneration/therapy , Stem Cell Transplantation , Aged , Aged, 80 and over , Cell- and Tissue-Based Therapy/methods , Female , Humans , Male , Middle Aged , Patients , Risk Factors , Surveys and QuestionnairesABSTRACT
Epithelial to mesenchymal transition (EMT) is a biological process involved in tissue morphogenesis and disease that causes dramatic changes in cell morphology, migration, proliferation, and gene expression. The retinal pigment epithelium (RPE), which supports the neural retina, can undergo EMT, producing fibrous epiretinal membranes (ERMs) associated with vision-impairing clinical conditions, such as macular pucker and proliferative vitreoretinopathy (PVR). We found that co-treatment with TGF-ß and TNF-α (TNT) accelerates EMT in adult human RPE stem cell-derived RPE cell cultures. We captured the global epigenomic and transcriptional changes elicited by TNT treatment of RPE and identified putative active enhancers associated with actively transcribed genes, including a set of upregulated transcription factors that are candidate regulators. We found that the vitamin B derivative nicotinamide downregulates these key transcriptional changes, and inhibits and partially reverses RPE EMT, revealing potential therapeutic routes to benefit patients with ERM, macular pucker and PVR.
Subject(s)
Epigenomics , Epiretinal Membrane/prevention & control , Epithelial-Mesenchymal Transition , Models, Biological , Niacinamide/therapeutic use , Retinal Pigment Epithelium/pathology , Stem Cells/metabolism , Transcriptome/genetics , Adult , Aged , Aged, 80 and over , Biomarkers/metabolism , Enhancer Elements, Genetic/genetics , Epiretinal Membrane/pathology , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation/drug effects , Humans , Middle Aged , Niacinamide/genetics , Niacinamide/pharmacology , Phenotype , Stem Cells/drug effects , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Transforming Growth Factor beta1/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Retinal pigment epithelial (RPE) cells are highly specialized neural cells that have several functions essential for vision. Progressive deterioration of RPE cells in elderly individuals can result in visual impairment and ultimately the blinding disease age-related macular degeneration. Subretinal transplantation of stem cell-derived RPE cell suspensions is being explored as a strategy to recover the damaged retina and improve vision. This approach may be improved by developing a vehicle that increases postinjection cell viability and distribution and integration of RPE cells. In this study, Food and Drug Administration-approved natural polymers, including alginate, methylcellulose, and hyaluronic acid (HA), were examined for performance as cell vehicles for adult human RPE stem cells (RPESCs). We compared the effect of RPESC storage as a cell suspension in these delivery vehicles for 1-96 hr at different temperatures on subsequent cell performance in a cell culture model. RPESC survival, attachment, distribution, proliferation, and differentiation into RPE cells were monitored by microscopy over the course of 8 weeks. Our in vitro results demonstrate that RPESC suspension in a 0.2% HA solution promotes better initial cell distribution, proliferation, cobblestone formation, and expression of RPE cell markers (microphthalmia-associated transcription factor and orthodenticle homeobox 2) after 96 hr of storage. These data suggest that HA addition to the vehicle can significantly enhance RPESC performance in vitro and is a promising strategy to pursue an improved delivery vehicle supporting in vivo RPE cell transplantation.
Subject(s)
Cell Culture Techniques , Cell Differentiation , Cells, Immobilized/metabolism , Preservation, Biological , Retinal Pigment Epithelium/metabolism , Stem Cells/metabolism , Aged , Cell Survival , Cells, Immobilized/cytology , Humans , Retinal Pigment Epithelium/cytology , Stem Cell Transplantation , Stem Cells/cytologyABSTRACT
Ocular regenerative therapies are on track to revolutionize treatment of numerous blinding disorders, including corneal disease, cataract, glaucoma, retinitis pigmentosa, and age-related macular degeneration. A variety of transplantable products, delivered as cell suspensions or as preformed 3D structures combining cells and natural or artificial substrates, are in the pipeline. Here we review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing. While recognizing the challenges, we look forward to a deep portfolio of sight-restoring, stem cell-based medicine. VIDEO ABSTRACT.
Subject(s)
Cornea/cytology , Eye Diseases/therapy , Regeneration , Retina/cytology , Stem Cells/cytology , Vision, Ocular , Animals , Eye Diseases/surgery , HumansABSTRACT
Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.
Subject(s)
Cell Transplantation/methods , Retinal Pigment Epithelium/transplantation , Transplantation, Heterologous/methods , Animals , Humans , Injections, Intraocular/methods , Macular Degeneration/therapy , Rats , Retina/transplantation , Retinal Pigment Epithelium/cytologyABSTRACT
Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.
Subject(s)
Adult Stem Cells/cytology , Cell Differentiation , Macular Degeneration/therapy , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/transplantation , Animals , Cell Culture Techniques , Cells, Cultured , Humans , Macular Degeneration/pathology , Rats , Retinal Pigment Epithelium/pathology , Swine , Vision, OcularABSTRACT
Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins, and those from the AMD donors show significantly increased complement and inflammatory factors, which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening, combined with transcriptome analysis, we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome, ribosome, and chromatin-modifying genes. Thus, targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD.
Subject(s)
Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Macular Degeneration/drug therapy , Macular Degeneration/metabolism , Niacinamide/therapeutic use , Cell Differentiation/drug effects , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Genotype , Humans , Immunohistochemistry , Retina/drug effects , Retina/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
PURPOSE: Assessing the morphologic properties of cells in microscopy images is an important task to evaluate cell health, identity, and purity. Typically, subjective visual assessments are accomplished by an experienced researcher. This subjective human step makes transfer of the evaluation process from the laboratory to the cell manufacturing facility difficult and time consuming. METHODS: Automated image analysis can provide rapid, objective measurements of cultured cells, greatly aiding manufacturing, regulatory, and research goals. Automated algorithms for classifying images based on appearance characteristics typically either extract features from the image and use those features for classification or use the images directly as input to the classification algorithm. In this study we have developed both feature and nonfeature extraction methods for automatically measuring "cobblestone" structure in human retinal pigment epithelial (RPE) cell cultures. RESULTS: A new approach using image compression combined with a Kolmogorov complexity-based distance metric enables robust classification of microscopy images of RPE cell cultures. The automated measurements corroborate determinations made by experienced cell biologists. We have also developed an approach for using steerable wavelet filters for extracting features to characterize the individual cellular junctions. CONCLUSIONS: Two image analysis techniques enable robust and accurate characterization of the cobblestone morphology that is indicative of viable RPE cultures for therapeutic applications.
Subject(s)
Cell Culture Techniques , Cell Shape , Image Interpretation, Computer-Assisted/methods , Retinal Pigment Epithelium/cytology , Stem Cells/cytology , Algorithms , Automation , Cells, Cultured , Epithelial-Mesenchymal Transition , Humans , Pattern Recognition, Automated/methodsABSTRACT
PURPOSE: Numerous preclinical studies have shown that transplantation of stem cell-derived retinal pigment epithelial cell (RPE) preserves photoreceptor cell anatomy in the dystrophic Royal College of Surgeons (RCS) rat. How rescue is spatially distributed over the eye, relative to the transplantation site, is less clear. To understand spatial variations in transplant efficacy, we have developed a method to measure the spatial distribution of rescued photoreceptor cells. METHODS: Human RPE Stem Cell-derived RPE (RPESC-RPE) cells were subretinally injected into RCS rat eyes. After tissue recovery and orientating the globe, a series of retinal sections were cut through the injected area. Sections were stained with DAPI (4',6-diamidino-2-phenylindole) and a number of photoreceptor nuclei were counted across the nasal-temporal and superior-inferior axes. These data were used to construct 2D maps of the area of photoreceptor cell saving. RESULTS: Photoreceptor cell preservation was detected in the injected temporal hemisphere and occupied areas greater than 4 mm(2) centered near the injection sites. Rescue was directed toward the central retina and superior and inferior poles, with maximal number of rescued photoreceptor cells proximal to the injection sites. CONCLUSIONS: RPESC-RPE transplantation preserves RCS photoreceptor cells. The photoreceptor cell contour maps readily convey the extent of rescue across the eye. The consistent alignment and quantification of results using this method allow the application of other downstream statistical analyses and comparisons to better understand transplantation therapy in the eye.
Subject(s)
Photoreceptor Cells, Vertebrate , Retinal Pigment Epithelium/cytology , Stem Cells , Animals , Humans , Rats , Rats, Long-Evans , Rats, Mutant StrainsABSTRACT
The retina, like other central nervous system tissues, has poor regenerative properties in humans. Therefore, diseases that cause retinal cell loss, such as Age-related macular degeneration (AMD), retinitis pigmentosa (RP), Leber congenital amaurosis, Usher syndrome, glaucoma, and diabetic retinopathy, typically result in permanent visual impairment. Stem cell technologies have revolutionized our ability to produce neural cells in abundant supply. Much stem cell research effort is focused on producing the required cell types for cell replacement, or to generate disease-in-a-dish models to elucidate novel disease mechanisms for therapeutic development. Here we review the recent advances in stem cell studies relevant to producing RPE and retinal cells, and highlight future directions.
Subject(s)
Eye Diseases/therapy , Regenerative Medicine/methods , Stem Cell Transplantation/methods , Cell Culture Techniques/methods , Cell Culture Techniques/trends , Cellular Reprogramming Techniques/methods , Cellular Reprogramming Techniques/trends , Embryonic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Regenerative Medicine/trends , Retinal Pigment Epithelium/cytology , Stem Cells/cytologyABSTRACT
The retinal pigment epithelium (RPE) is a pigmented cellular monolayer that supports photoreceptor cells located in the overlying neural retina. The RPE is critical for vision and its dysfunction results in numerous pathologies, several with limited available disease-altering strategies. Regeneration of the retina from RPE is robust in lower vertebrates, but is not normally exhibited in mammals. We recently found that a subpopulation of human RPE cells can be stimulated in culture to generate multipotent self-renewing cells-the RPE stem cell (RPESC). RPESC can be expanded to generate RPE progeny that are a potential source for cell replacement therapy. Alternatively, RPESC can produce mesenchymal progeny which serve as a disease model of epiretinal membrane formation. Yet another potential application of RPESCs is activation within the eye to awaken dormant endogenous repair.
Subject(s)
Cell Differentiation , Cell Proliferation , Retinal Pigment Epithelium/cytology , Stem Cells/cytology , Animals , Cell Culture Techniques , Cell Lineage , Cells, Cultured , Humans , Multipotent Stem Cells/cytology , Stem Cell Transplantation/methodsABSTRACT
Transient expression of exogenous proteins facilitates studies of molecular mechanisms and utility for transplantation of retinal pigment epithelial (RPE) cells in culture. Here, we compared expression of the membrane protein ß5 integrin-GFP (ß5-GFP) in two recently established models of differentiated human RPE, adult RPE stem cell-derived RPE and primary fetal RPE, upon infection with recombinant adenovirus or transfection with DNA in liposomes. We varied viral titer and duration of virus incubation and examined ß5-GFP and the tight junction marker ZO-1 in manipulated cells by confocal microscopy. Fewer than 5 % of cells expressed ß5-GFP after liposome-mediated transfection. The percentage of cells with detectable ß5-GFP exceeded 90 % after adenovirus infection for as little as 1 h. Decreasing virus titer two-fold did not alter the fraction of cells expressing ß5-GFP but increased variability of ß5-GFP level among cells. In cells with low expression levels, ß5-GFP localized mostly to the apical plasma membrane like endogenous αvß5 integrin. In cells with high expression levels, ß5-GFP localized to the cytoplasm in addition to the apical surface suggesting accumulation in trafficking compartments. Altogether, adenovirus delivery yields efficient exogenous membrane protein expression of correct polarity in differentiated human RPE cells in culture.
Subject(s)
Adenoviridae/genetics , Membrane Proteins/genetics , Pigment Epithelium of Eye/metabolism , Cell Polarity , Cells, Cultured , Epithelial Cells/metabolism , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Integrin beta Chains/genetics , Integrin beta Chains/metabolism , Liposomes , Membrane Proteins/metabolism , Microscopy, Confocal , Pigment Epithelium of Eye/cytology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reproducibility of Results , Transfection/methodsABSTRACT
PURPOSE: We tested what native features have been preserved with a new culture protocol for adult human RPE. METHODS: We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used. RESULTS: Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 µM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 µL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01). CONCLUSIONS: The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.
Subject(s)
Eye Proteins/metabolism , Nerve Growth Factors/metabolism , Retinal Pigment Epithelium/metabolism , Stem Cells/metabolism , Adult , Aged , Aged, 80 and over , Cell Polarity , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Humans , Immunoblotting , Immunohistochemistry , Microscopy, Electron , Middle Aged , Retinal Pigment Epithelium/ultrastructure , Stem Cells/ultrastructureABSTRACT
Transplantation of the retinal pigment epithelium (RPE) is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC) as an alternative RPE source. Polarized monolayers of adult hRPESC-derived RPE grown on polyester (PET) membranes had near-native characteristics. Trephined pieces of RPE monolayers on PET were transplanted subretinally in the rabbit, a large-eyed animal model. After 4 days, retinal edema was observed above the implant, detected by spectral domain optical coherence tomography (SD-OCT) and fundoscopy. At 1 week, retinal atrophy overlying the fetal or adult transplant was observed, remaining stable thereafter. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. Taken together, the xeno-RPE survived with retained characteristics in the subretinal space. These experiments support that adult hRPESC-derived RPE are a potential source for transplantation therapies.
Subject(s)
Polyesters/chemistry , Retinal Pigment Epithelium/cytology , Stem Cells/cytology , Aged , Animals , Cells, Cultured , Female , Humans , Papilledema/diagnostic imaging , Papilledema/pathology , Rabbits , Radiography , Retina/pathology , Stem Cell Transplantation , Stem Cells/metabolism , Tomography, Optical Coherence , Transplantation, HeterologousABSTRACT
An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings.
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation/drug effects , Fibroblast Growth Factor 2/pharmacology , Stem Cells/cytology , Animals , Cells, Cultured , Cells, Immobilized/cytology , Cells, Immobilized/drug effects , Culture Media/pharmacology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/enzymology , Enzyme Activation/drug effects , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/enzymology , Lactic Acid , MAP Kinase Signaling System/drug effects , Mice , Microspheres , Mitogen-Activated Protein Kinases/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/drug effects , Neural Stem Cells/enzymology , Polyglycolic Acid , Polylactic Acid-Polyglycolic Acid Copolymer , Stem Cells/drug effects , Stem Cells/enzymologyABSTRACT
PURPOSE: The goal of this study was to examine changes in the expression of transcripts and proteins associated with drusen in Age-related Macular Degeneration (AMD) after exposing human retinal pigment epithelium (hRPE) cells to chronic oxidative stress. METHODS: Primary adult human RPE cells were isolated from cadaveric donor eyes. The subpopulation of RPE stem cells (RPESCs) was activated, expanded, and then differentiated into RPE progeny. Confluent cultures of RPESC-derived hRPE and ARPE-19 cells were exposed to a regimen of tert-butylhydroperoxide (TBHP) for 1-5 days. After treatment, gene expression was measured by quantitative PCR (qPCR), protein expression was assessed by immunocytochemistry and transepithelial resistance and cell toxicity were measured. RESULTS: hRPE cells exposed to a regimen of TBHP for 5 days upregulate expression of several molecules identified in drusen, including molecular chaperones and pro-angiogenic factors. 5-day TBHP treatment was significantly more effective than 1-day treatment at eliciting these effects. The extent of hRPE response to 5-day treatment varied significantly between individual donors, nevertheless, 6 transcripts were reliably significantly upregulated. ARPE-19 cells treated with the same 5-day stress regime did not show the same pattern of response and did not upregulate this group of transcripts. CONCLUSIONS: RPESC-derived hRPE cells change significantly when exposed to repeated oxidative stress conditions, upregulating expression of several drusen-related proteins and transcripts. This is consistent with the hypothesis that hRPE cells are competent to be a source of proteins found in drusen deposits. Our results suggest that donor-specific genetic and environmental factors influence the RPE stress response. ARPE-19 cells appear to be less representative of AMD-like changes than RPESC-derived hRPE. This adult stem cell-based system using chronic TBHP treatment of hRPE represents a novel in vitro model useful for the study of drusen formation and dry AMD pathophysiology.