A fluorophore-conjugated reagent enabling rapid detection, isolation and live tracking of senescent cells.

Cellular senescence is a stress-response mechanism implicated in various physiological processes, diseases, and aging. Current detection approaches have partially addressed the issue of senescent cell identification in clinical specimens. Effective methodologies enabling precise isolation or live tracking of senescent cells are still lacking. In-depth analysis of truly senescent cells is, therefore, an extremely challenging task. We report (1) the synthesis and validation of a fluorophore-conjugated, Sudan Black-B analog (GLF16), suitable for in vivo and in vitro analysis of senescence by fluorescence microscopy and flow cytometry and (2) the development and application of a GLF16-carrying micelle vector facilitating GLF16 uptake by living senescent cells in vivo and in vitro. The compound and the applied methodology render isolation of senescent cells an easy, rapid, and precise process. Straightforward nanocarrier-mediated GLF16 delivery in live senescent cells comprises a unique tool for characterization of senescence at an unprecedented depth.

Enantiomeric separation of nefopam and cathinone derivatives using a supramolecular deep eutectic solvent as a chiral selector in capillary electrophoresis.

The present study investigates the utilization of a supramolecular deep eutectic solvent (SUPRADES), consisting of sulfated-ß-cyclodextrin (S-ß-CD) and citric acid (CA), as a chiral selector (CS) in capillary electrophoresis for the enantiomeric separation of nefopam (NEF) and five cathinone derivatives (3-methylmethcathinone [3-MMC], 4-methylmethcathinone [4-MMC], 3,4-dimethylmethcathinone [3,4-DMMC], 4-methylethcathinone [4-MEC], and 3,4-methylendioxycathinone [MDMC]). A significant improvement in enantiomeric separation of the target analytes was observed upon the addition of S-ß-CD-CA to the background electrolyte (BGE), leading to a baseline separation of all analytes. In particular, the optimum percentage of S-ß-CD-CA, added to the BGE, was determined to be 0.075% v/v for NEF (Rs = 1.5) and 0.050% v/v for three out of five cathinone derivatives (Rs = 1.5, 1.6, and 2.4 for 3-MMC, 4-MEC, and 3,4-DMMC, respectively). In the case of 4-MMC and MDMC, a higher percentage of the CS, equal to 0.075% and 0.10% v/v, respectively, was required to achieve baseline separation (Rs = 1.5, 1.9 for MDMC and 4-MMC, respectively). The outcomes of the present study highlight the potential effectiveness of using SUPRADES as a CS in electrophoretic enantioseparations.

Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids.

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina.

Diabetic retinopathy and the role of Omega-3 PUFAs: A narrative review.

Diabetes mellitus has been a major cause of concern for the past few decades. As the number of diabetic patients increases, so too does the occurrence of its complications. Diabetic retinopathy (DR) is one of these and constitutes the most common cause of blindness amongst working-age individuals. Chronic exposure to a hyperglycaemic environment remains the driving force of a cascade of molecular events that disrupt the microvasculature of the retina and if left untreated can lead to blindness. In this review, we identify oxidative stress as a major implication in the pathway to the development of DR and speculate that it plays a central role especially in the early stages of the disease. Cells lose their antioxidant capacity under a hyperglycaemic state, free radicals are formed and eventually apoptosis ensues. The polyol pathway; advanced glycation end-product formation; the protein kinase C pathway, and the hexosamine pathway are found to contribute to the increase in oxidative stress observed in diabetic patients. We also investigate the use of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in DR. These molecules possess antioxidant and anti-inflammatory properties and have been previously investigated for use in other ocular pathologies with promising results. In this review we present the latest findings in pre-clinical and clinical studies for the use of ω-3 PUFAs in DR. We hypothesise that ω-3 PUFAs could be beneficial for DR in ways of reducing the oxidative stress and limiting the progression of the disease that threatens the eyesight of the patient, in conjunction with conventional therapy.

Direct transcriptomic comparison of xenobiotic metabolism and toxicity pathway induction of airway epithelium models at an air-liquid interface generated from induced pluripotent stem cells and primary bronchial epithelial cells.

The airway epithelium represents the main barrier between inhaled air and the tissues of the respiratory tract and is therefore an important point of contact with xenobiotic substances into the human body. Several studies have recently shown that in vitro models of the airway grown at an air-liquid interface (ALI) can be particularly useful to obtain mechanistic information about the toxicity of chemical compounds. However, such methods are not very amenable to high throughput since the primary cells cannot be expanded indefinitely in culture to obtain a sustainable number of cells. Induced pluripotent stem cells (iPSCs) have become a popular option in the recent years for modelling the airways of the lung, but despite progress in the field, such models have so far not been assessed for their ability to metabolise xenobiotic compounds and how they compare to the primary bronchial airway model (pBAE). Here, we report a comparative analysis by TempoSeq (oligo-directed sequencing) of an iPSC-derived airway model (iBAE) with a primary bronchial airway model (pBAE). The iBAE and pBAE were differentiated at an ALI and then evaluated in a 5-compound screen with exposure to a sub-lethal concentration of each compound for 24 h. We found that despite lower expression of xenobiotic metabolism genes, the iBAE similarly predicted the toxic pathways when compared to the pBAE model. Our results show that iPSC airway models at ALI show promise for inhalation toxicity assessments with further development.

SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells.

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs.

Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent.

Induced pluripotent stem cell (iPSC)-derived retinal organoids provide a platform to study human retinogenesis, disease modeling, and compound screening. Although retinal organoids may represent tissue structures with greater physiological relevance to the in vivo human retina, their generation is not without limitations. Various protocols have been developed to enable development of organoids with all major retinal cell types; however, variability across iPSC lines is often reported. Modulating signaling pathways important for eye formation, such as those involving bone morphogenetic protein 4 (BMP4) and insulin-like growth factor 1 (IGF1), is a common approach used for the generation of retinal tissue in vitro. We used three human iPSC lines to generate retinal organoids by activating either BMP4 or IGF1 signaling and assessed differentiation efficiency by monitoring morphological changes, gene and protein expression, and function. Our results showed that the ability of iPSC to give rise to retinal organoids in response to IGF1 and BMP4 activation was line- and method-dependent. This demonstrates that careful consideration is needed when choosing a differentiation approach, which would also depend on overall project aims.

A prototype PET/SPECT/X-rays scanner dedicated for whole body small animal studies.

OBJECTIVE: To present a prototype tri-modal imaging system, consisting of a single photon emission computed tomography (SPET), a positron emission tomography (PET), and a computed tomography (CT) subsystem, evaluated in planar mode. MATERIALS AND METHODS: The subsystems are mounted on a rotating gantry, so as to be able to allow tomographic imaging in the future. The system, designed and constructed by our group, allows whole body mouse imaging of competent performance and is currently, to the best of our knowledge, unequaled in a national and regional level. The SPET camera is based on two Position Sensitive Photomultiplier Tubes (PSPMT), coupled to a pixilated Sodium Iodide activated with Thallium (NaI(Tl)) scintillator, having an active area of 5x10cm2. The dual head PET camera is also based on two pairs of PSPMT, coupled to pixelated berillium germanium oxide (BGO) scintillators, having an active area of 5x10cm2. The X-rays system consists of a micro focus X-rays tube and a complementary metal-oxide-semiconductor (CMOS) detector, having an active area of 12x12cm2. RESULTS: The scintigraphic mode has a spatial resolution of 1.88mm full width at half maximum (FWHM) and a sensitivity of 107.5cpm/0.037MBq at the collimator surface. The coincidence PET mode has an average spatial resolution of 3.5mm (FWHM) and a peak sensitivity of 29.9cpm/0.037MBq. The X-rays spatial resolution is 3.5lp/mm and the contrast discrimination function value is lower than 2%. CONCLUSION: A compact tri-modal system was successfully built and evaluated for planar mode operation. The system has an efficient performance, allowing accurate and informative anatomical and functional imaging, as well as semi-quantitative results. Compared to other available systems, it provides a moderate but comparable performance, at a fraction of the cost and complexity. It is fully open, scalable and its main purpose is to support groups on a national and regional level and provide an open technological platform to study different detector components and acquisition strategies.

Hit-to-Lead Optimization of Heterocyclic Carbonyloxycarboximidamides as Selective Antagonists at Human Adenosine A3 Receptor.

Antagonism of the human adenosine A3 receptor (hA3R) has potential therapeutic application. Alchemical relative binding free energy calculations of K18 and K32 suggested that the combination of a 3-(2,6-dichlorophenyl)-isoxazolyl group with 2-pyridinyl at the ends of a carbonyloxycarboximidamide group should improve hA3R affinity. Of the 25 new analogues synthesized, 37 and 74 showed improved hA3R affinity compared to K18 (and K32). This was further improved through the addition of a bromine group to the 2-pyridinyl at the 5-position, generating compound 39. Alchemical relative binding free energy calculations, mutagenesis studies and MD simulations supported the compounds' binding pattern while suggesting that the bromine of 39 inserts deep into the hA3R orthosteric pocket, so highlighting the importance of rigidification of the carbonyloxycarboximidamide moiety. MD simulations highlighted the importance of rigidification of the carbonyloxycarboximidamide, while suggesting that the bromine of 39 inserts deep into the hA3R orthosteric pocket, which was supported through mutagenesis studies 39 also selectively antagonized endogenously expressed hA3R in nonsmall cell lung carcinoma cells, while pharmacokinetic studies indicated low toxicity enabling in vivo evaluation. We therefore suggest that 39 has potential for further development as a high-affinity hA3R antagonist.

Retinal cells derived from patients with DRAM2-dependent CORD21 dystrophy exhibit key lysosomal enzyme deficiency and lysosomal content accumulation.

Biallelic mutations in DRAM2 lead to an autosomal recessive cone-rod dystrophy known as CORD21, which typically presents between the third and sixth decades of life. Although DRAM2 localizes to the lysosomes of photoreceptor and retinal pigment epithelium (RPE) cells, its specific role in retinal degeneration has not been fully elucidated. In this study, we generated and characterized retinal organoids (ROs) and RPE cells from induced pluripotent stem cells (iPSCs) derived from two CORD21 patients. Our investigation revealed that CORD21-ROs and RPE cells exhibit abnormalities in lipid metabolism, defects in autophagic flux, accumulation of aberrant lysosomal content, and reduced lysosomal enzyme activity. We identified potential interactions of DRAM2 with vesicular trafficking proteins, suggesting its involvement in this cellular process. These findings collectively suggest that DRAM2 plays a crucial role in maintaining the integrity of photoreceptors and RPE cells by regulating lysosomal function, autophagy, and potentially vesicular trafficking.

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16.

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.1.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Synthesis, cytotoxic activity evaluation and mechanistic investigation of novel 3,7-diarylsubstituted 6-azaindoles.

A number of new disubstituted 6-azaindoles have been designed and synthesized bearing a crucial structural modification in respect to an analogous antiproliferative hit compound. The synthesis was performed using 2-amino-3-nitro-4-picoline, that was suitably modified and converted to 7-chloro-3-iodo-6-azaindole and this central scaffold was used for successive Suzuki-type couplings, to result in the target compounds. The evaluation of the cytotoxic activity was performed against four human cancer cell lines, as well as a normal human fibroblast strain. Certain compounds possessed strong anticancer activity without affecting normal cells. At subcytotoxic concentrations for cancer cells, these compounds displayed an anti-proliferative effect by arresting the cells at the G2/M phase of the cell cycle, which could be associated with the observed decrease in the phosphorylation levels of the MEK1- ERK1/2 pathway and/or the activation of the p53-p21WAF1 axis.

Novel H2S-Releasing Bifunctional Antihistamine Molecules with Improved Antipruritic and Reduced Sedative Actions.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter with anti-inflammatory actions that also reduces itching. To test whether a combination of an antihistamine with a H2S donor has improved antipruritic efficacy, bifunctional molecules with antihistamine and H2S-releasing pharmacophores were synthesized and tested in vitro and in vivo. H2S release from the hybrid molecules was evaluated with the methylene blue and lead acetate methods, and H1-blocking activity was assessed by determining tissue factor expression inhibition. All new compounds released H2S in a dose-dependent manner and retained histamine blocking activity. Two compounds with the highest potency were evaluated in vivo for their antipruritic as well as sedative action; they proved to possess higher efficacy in inhibiting histamine-induced pruritus and decreased sedative effects compared to the parent compounds (hydroxyzine and cetirizine), suggesting that they exhibit superior antipruritic action and limited side effects that likely arise from the H2S-releasing moiety.

Discovery of New 1,4,6-Trisubstituted-1H-pyrazolo[3,4-b]pyridines with Anti-Tumor Efficacy in Mouse Model of Breast Cancer.

Purine analogues are important therapeutic tools due to their affinity to enzymes or receptors that are involved in critical biological processes. In this study, new 1,4,6-trisubstituted pyrazolo[3,4-b]pyridines were designed and synthesized, and their cytotoxic potential was been studied. The new derivatives were prepared through suitable arylhydrazines, and upon successive conversion first to aminopyrazoles, they were converted then to 1,6-disubstituted pyrazolo[3,4-b]pyridine-4-ones; this served as the starting point for the synthesis of the target compounds. The cytotoxic activity of the derivatives was evaluated against several human and murine cancer cell lines. Substantial structure activity relationships (SARs) could be extracted, mainly concerning the 4-alkylaminoethyl ethers, which showed potent in vitro antiproliferative activity in the low µM level (0.75-4.15 µΜ) without affecting the proliferation of normal cells. The most potent analogues underwent in vivo evaluation and were found to inhibit tumor growth in vivo in an orthotopic breast cancer mouse model. The novel compounds exhibited no systemic toxicity; they affected only the implanted tumors and did not interfere with the immune system of the animals. Our results revealed a very potent novel compound which could be an ideal lead for the discovery of promising anti-tumor agents, and could also be further explored for combination treatments with immunotherapeutic drugs.

Hydrolytic Activity of Mitochondrial F1FO-ATP Synthase as a Target for Myocardial Ischemia-Reperfusion Injury: Discovery and In Vitro and In Vivo Evaluation of Novel Inhibitors.

F1FO-ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, it reverses its activity, hydrolyzing ATP and leading to energetic deficit and cardiac injury. We aimed to discover novel inhibitors of ATP hydrolysis, accessing the druggability of the target within ischemia(I)/reperfusion(R) injury. New molecular scaffolds were revealed using ligand-based virtual screening methods. Fifty-five compounds were tested on isolated murine heart mitochondria and H9c2 cells for their inhibitory activity. A pyrazolo[3,4-c]pyridine hit structure was identified and optimized in a hit-to-lead process synthesizing nine novel derivatives. Three derivatives significantly inhibited ATP hydrolysis in vitro, while in vivo, they reduced myocardial infarct size (IS). The novel compound 31 was the most effective in reducing IS, validating that inhibition of F1FO-ATP hydrolytic activity can serve as a target for cardioprotection during ischemia. Further examination of signaling pathways revealed that the cardioprotection mechanism is related to the increased ATP content in the ischemic myocardium and increased phosphorylation of PKA and phospholamban, leading to the reduction of apoptosis.

Human Retinal Organoids Provide a Suitable Tool for Toxicological Investigations: A Comprehensive Validation Using Drugs and Compounds Affecting the Retina.

Retinal drug toxicity screening is essential for the development of safe treatment strategies for a large number of diseases. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to the human retina and the ease of generation in large-scale formats. In this study, two hPSC cell lines were differentiated to retinal organoids, which comprised all key retinal cell types in multiple nuclear and synaptic layers. Single-cell RNA-Seq of retinal organoids indicated the maintenance of retinal ganglion cells and development of bipolar cells: both cell types segregated into several subtypes. Ketorolac, digoxin, thioridazine, sildenafil, ethanol, and methanol were selected as key compounds to screen on retinal organoids because of their well-known retinal toxicity profile described in the literature. Exposure of the hPSC-derived retinal organoids to digoxin, thioridazine, and sildenafil resulted in photoreceptor cell death, while digoxin and thioridazine additionally affected all other cell types, including Müller glia cells. All drug treatments caused activation of astrocytes, indicated by dendrites sprouting into neuroepithelium. The ability to respond to light was preserved in organoids although the number of responsive retinal ganglion cells decreased after drug exposure. These data indicate similar drug effects in organoids to those reported in in vivo models and/or in humans, thus providing the first robust experimental evidence of their suitability for toxicological studies.

Conjunctival epithelial cells resist productive SARS-CoV-2 infection.

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.

Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient-induced pluripotent stem cell-derived retinal pigment epithelium cells.

INTRODUCTION: Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. METHODS: We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. RESULTS: To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. CONCLUSIONS: Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.

Retinal pigment epithelium extracellular vesicles are potent inducers of age-related macular degeneration disease phenotype in the outer retina.

Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.