Contribution of extracellular vesicles for the pathogenesis of retinal diseases: shedding light on blood-retinal barrier dysfunction.

Retinal degenerative diseases, including diabetic retinopathy (DR) and age-related macular degeneration (AMD), loom as threats to vision, causing detrimental effects on the structure and function of the retina. Central to understanding these diseases, is the compromised state of the blood-retinal barrier (BRB), an effective barrier that regulates the influx of immune and inflammatory components. Whether BRB breakdown initiates retinal distress, or is a consequence of disease progression, remains enigmatic. Nevertheless, it is an indication of retinal dysfunction and potential vision loss.The intricate intercellular dialogues among retinal cell populations remain unintelligible in the complex retinal milieu, under conditions of inflammation and oxidative stress. The retina, a specialized neural tissue, sustains a ceaseless demand for oxygen and nutrients from two vascular networks. The BRB orchestrates the exchange of molecules and fluids within this specialized region, comprising the inner BRB (iBRB) and the outer BRB (oBRB). Extracellular vesicles (EVs) are small membranous structures, and act as messengers facilitating intercellular communication in this milieu.EVs, both from retinal and peripheral immune cells, increase complexity to BRB dysfunction in DR and AMD. Laden with bioactive cargoes, these EVs can modulate the retinal microenvironment, influencing disease progression. Our review delves into the multifaceted role of EVs in retinal degenerative diseases, elucidating the molecular crosstalk they orchestrate, and their microRNA (miRNA) content. By shedding light on these nanoscale messengers, from their biogenesis, release, to interaction and uptake by target cells, we aim to deepen the comprehension of BRB dysfunction and explore their therapeutic potential, therefore increasing our understanding of DR and AMD pathophysiology.

Regenerative treatment of ophthalmic diseases with stem cells: Principles, progress, and challenges.

Background: Degenerate eye disorders, such as glaucoma, cataracts and age-related macular degeneration (AMD), are prevalent causes of blindness and visual impairment worldwide. Other eye disorders, including limbal stem cell deficiency (LSCD), dry eye diseases (DED), and retinitis pigmentosa (RP), result in symptoms such as ocular discomfort and impaired visual function, significantly impacting quality of life. Traditional therapies are limited, primarily focus on delaying disease progression, while emerging stem cell therapy directly targets ocular tissues, aiming to restore ocular function by reconstructing ocular tissue. Main text: The utilization of stem cells for the treatment of diverse degenerative ocular diseases is becoming increasingly significant, owing to the regenerative and malleable properties of stem cells and their functional cells. Currently, stem cell therapy for ophthalmopathy involves various cell types, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and retinal progenitor cells (RPCs). In the current article, we will review the current progress regarding the utilization of stem cells for the regeneration of ocular tissue covering key eye tissues from the cornea to the retina. These therapies aim to address the loss of functional cells, restore damaged ocular tissue and or in a paracrine-mediated manner. We also provide an overview of the ocular disorders that stem cell therapy is targeting, as well as the difficulties and opportunities in this field. Conclusions: Stem cells can not only promote tissue regeneration but also release exosomes to mitigate inflammation and provide neuroprotection, making stem cell therapy emerge as a promising approach for treating a wide range of eye disorders through multiple mechanisms.

Small extracellular vesicles of organoid-derived human retinal stem cells remodel Müller cell fate via miRNA: A novel remedy for retinal degeneration.

Remodeling retinal Müller glial fate, including gliosis inhibition and pro-reprogramming, represents a crucial avenue for treating degenerative retinal diseases. Stem cell transplantation exerts effects on modulating retinal Müller glial fate. However, the optimized stem cell products and the underlying therapeutic mechanisms need to be investigated. In the present study, we found that retinal progenitor cells from human embryonic stem cell-derived retinal organoids (hERO-RPCs) transferred extracellular vesicles (EVs) into Müller cells following subretinal transplantation into RCS rats. Small EVs from hERO-RPCs (hERO-RPC-sEVs) were collected and were found to delay photoreceptor degeneration and protect retinal function in RCS rats. hERO-RPC-sEVs were taken up by Müller cells both in vivo and in vitro, and inhibited gliosis while promoting early dedifferentiation of Müller cells. We further explored the miRNA profiles of hERO-RPC-sEVs, which suggested a functional signature associated with neuroprotection and development, as well as the regulation of stem cell and glial fate. Mechanistically, hERO-RPC-sEVs might regulate the fate of Müller cells by miRNA-mediated nuclear factor I transcription factors B (NFIB) downregulation. Collectively, our findings offer novel mechanistic insights into stem cell therapy and promote the development of EV-centered therapeutic strategies.

Compounding engineered mesenchymal stem cell-derived exosomes: A potential rescue strategy for retinal degeneration.

The prevalence of retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, has been increasing globally and is linked to the aging population and improved life expectancy. These diseases are characterized by chronic, progressive neuronal damage or depletion of the photoreceptor cells in the retina, and limited effective treatment options are currently available. Mesenchymal stem cell-derived exosomes (MSC-EXOs) containing cytokines, growth factors, lipids, mRNA, and miRNA, which act as mediators of intercellular communication transferring bioactive molecules to recipient cells, offer an appealing, non-cellular nanotherapeutic approach for retinal degenerative diseases. However, treatment specificity is compromised due to their high heterogeneity in size, content, functional effects, and parental cellular source. To improve this, engineered MSC-EXOs with increased drug-loading capacity, targeting ability, and resistance to bodily degradation and elimination have been developed. This review summarizes the recent advances in miRNAs of MSC-EXOs as a treatment for retinal degeneration, discussing the strategies and methods for engineering therapeutic MSC-EXOs. Notably, to address the single functional role of engineered MSC-EXOs, we propose a novel concept called "Compound Engineered MSC-EXOs (Co-E-MSC-EXOs)" along with its derived potential therapeutic approaches. The advantages and challenges of employing Co-E-MSC-EXOs for retinal degeneration in clinical applications, as well as the strategies and issues related to them, are also highlighted.

Regulated Necrosis in Glaucoma: Focus on Ferroptosis and Pyroptosis.

Glaucoma is one of the most common causes of irreversible blindness worldwide. This neurodegenerative disease is characterized by progressive and irreversible damage to retinal ganglion cells (RGCs) and optic nerves, which can lead to permanent loss of peripheral and central vision. To date, maintaining long-term survival of RGCs using traditional treatments, such as medication and surgery, remains challenging, as these do not promote optic nerve regeneration. Therefore, it is of great clinical and social significance to investigate the mechanisms of optic nerve degeneration in depth and find reliable targets to provide pioneering methods for the prevention and treatment of glaucoma. Regulated necrosis is a form of genetically programmed cell death associated with the maintenance of homeostasis and disease progression in vivo. An increasing body of innovative evidence has recognized that aberrant activation of regulated necrosis pathways is a common feature in neurodegenerative diseases, such as Alzheimer's, Parkinson's, and glaucoma, resulting in unwanted loss of neuronal cells and function. Among them, ferroptosis and pyroptosis are newly discovered forms of regulated cell death actively involved in the pathophysiological processes of RGCs loss and optic nerve injury. This was shown by a series of in vivo and in vitro studies, and these mechanisms have been emerging as a key new area of scientific research in ophthalmic diseases. In this review, we focus on the molecular mechanisms of ferroptosis and pyroptosis and their regulatory roles in the pathogenesis of glaucoma, with the aim of exploring their implications as potential therapeutic targets and providing new perspectives for better clinical decision-making in glaucoma treatment.

Human Retinal Organoids in Therapeutic Discovery: A Review of Applications.

Human embryonic stem cells (hESCs)- and induced pluripotent stem cells (hiPSCs)-derived retinal organoids (ROs) are three-dimensional laminar structures that recapitulate the developmental trajectory of the human retina. The ROs provide a fascinating tool for basic science research, eye disease modeling, treatment development, and biobanking for tissue/cell replacement. Here we review the previous studies that paved the way for RO technology, the two most widely accepted, standardized protocols to generate ROs, and the utilization of ROs in medical discovery. This review is conducted from the perspective of basic science research, transplantation for regenerative medicine, disease modeling, and therapeutic development for drug screening and gene therapy. ROs have opened avenues for new technologies such as assembloids, coculture with other organoids, vasculature or immune cells, microfluidic devices (organ-on-chip), extracellular vesicles for drug delivery, biomaterial engineering, advanced imaging techniques, and artificial intelligence (AI). Nevertheless, some shortcomings of ROs currently limit their translation for medical applications and pose a challenge for future research. Despite these limitations, ROs are a powerful tool for functional studies and therapeutic strategies for retinal diseases.

Retinoic Acid Receptor-Related Orphan Receptors (RORs) in Eye Development and Disease.

The retinoic acid receptor-related orphan receptors (RORs) are ligand-mediated transcription factors with important biological roles in regulating circadian rhythms, metabolism, immunity, angiogenesis, inflammation, and development. They belong to the superfamily of nuclear receptors and include three family members: RORα, RORß, and RORγ. Currently identified ROR ligands include cholesterol and cholesterol derivatives for RORα and RORγ, and stearic acid and all-trans retinoic acid for RORß. Aberrant signaling of the RORs is involved in the pathogenesis of several human diseases including autoimmune diseases, metabolic disorders, and certain cancers. In the eye, RORs regulate normal development of the lens and the retina, and also contribute to potentially blinding eye diseases, especially retinal vascular diseases. Here, we review the role of RORs in eye development and disease to highlight their potential as druggable targets for therapeutic development in retinal vascular and degenerative diseases.

An Engineering Platform for Clinical Application of Optogenetic Therapy in Retinal Degenerative Diseases.

Optogenetics is a new approach for controlling neural circuits with numerous applications in both basic and clinical science. In retinal degenerative diseases, the photoreceptors die, but inner retinal cells remain largely intact. By expressing light sensitive proteins in the remaining cells, optogenetics has the potential to offer a novel approach to restoring vision. In the past several years, optogenetics has advanced into an early clinical stage, and promising results have been reported. At the current stage, there is an urgent need to develop hardware and software for clinical training, testing, and rehabilitation in optogenetic therapy, which is beyond the capability of existing ophthalmic equipment. In this paper, we present an engineering platform consisting of hardware and software utilities, which allow clinicians to interactively work with patients to explore and assess their vision in optogenetic treatment, providing the basis for prosthetic design, customization, and prescription. This approach is also applicable to other therapies that utilize light activation of neurons, such as photoswitches.Clinical and Translational Impact Statement-The engineering platform allows clinicians to conduct training, testing, and rehabilitation in optogenetic gene therapy for retinal degenerative diseases, providing the basis for prosthetic design, customization, and prescription.

Targeting Necroptosis: A Novel Therapeutic Option for Retinal Degenerative Diseases.

The discovery of the necroptosis, a form of regulated necrosis that is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like pseudokinase (MLKL), represents a major breakthrough that has dramatically altered the conception of necrosis - traditionally thought of as uncontrolled cell death - in various human diseases. Retinal cell death is a leading cause of blindness and has been identified in most retinal diseases, e.g., age-related macular degeneration, glaucoma, retinal detachment, retinitis pigmentosa, etc. Increasing evidence demonstrates that retinal degenerative diseases also share a common mechanism in necroptosis. Exacerbated necroptotic cell death hinders the treatment for retinal degenerative diseases. In this review, we highlight recent advances in identifying retinal necroptosis, summarize the underlying mechanisms of necroptosis in retinal degenerative diseases, and discuss potential anti-necroptosis strategies, such as selective inhibitors and chemical agents, for treating retinal degenerative diseases.

Retinal Tissue Engineering: Regenerative and Drug Delivery Approaches.

In recent decades, the improvement of photoreceptor-cell transplantation has been used as an effective therapeutic approach to treat retinal degenerative diseases. In this review, the effect of different factors on the differentiation process and stem cells toward photoreceptors along with cell viability, morphology, migration, adhesion, proliferation, and differentiation efficiency is discussed. Scientists are researching to better recognize the reasons for retinal degeneration, as well as discovering novel therapeutic methods to restore lost vision. In this field, several procedures and treatments in the implantation of stem cells-derived retinal cells have been explored for clinical trials. However, the number of these clinical trials is too small to draw sound decisions about whether stem-cell therapies can offer a cure for retinal diseases. Nevertheless, future research directions have started for patients affected by retinal degeneration and promising findings have been obtained.

Hydrogels to Support Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

PURPOSE: Retinal pigment epithelial (RPE) cells are highly specialized neural cells with several functions essential for vision. Progressive deterioration of RPE cells in elderly individuals can result in visual impairment and, ultimately, blinding disease. While human embryonic stem cell-derived RPE cell (hESC-RPE) growth conditions are generally harsher than those of cell lines, the subretinal transplantation of hESC-RPE is being clinically explored as a strategy to recover the damaged retina and improve vision. The cell-adhesion ability of the support is required for RPE transplantation, where pre-polarized cells can maintain specific functions on the scaffold. This work examined four typical biodegradable hydrogels as supports for hESC-RPE growth. METHODS: Four biodegradable hydrogels were examined: gelatin methacryloyl (GelMA), hyaluronic acid methacryloyl (HAMA), alginate, and fibrin hydrogels. ARPE-19 and hESC-RPE cells were seeded onto the hydrogels separately, and the ability of these supports to facilitate adherence, proliferation, and homogeneous distribution of differentiated hESC-RPE cells was investigated. Furthermore, the hydrogel's subretinal bio-compatibility was assessed in vivo. RESULTS: We showed that ARPE-19 and hESC-RPE cells adhered and proliferated only on the fibrin support. The monolayer formed when cells reached confluency, demonstrating the polygonal semblance, and revealing actin filaments that moved along the cytoplasm. The expression of tight junction proteins at cell interfaces on the 14th day of seeding demonstrated the barrier function of epithelial cells on polymeric surfaces and the interaction between cells. Moreover, the expression of proteins crucial for retinal functions and matrix production was positively affected by fibrin, with an increment of PEDF. Our in vivo investigation with fibrin hydrogels revealed high short-term subretinal biocompatibility. CONCLUSIONS: The research of stem cell-based cell therapy for retinal degenerative diseases is more complicated than that of cell lines. Our results showed that fibrin is a suitable scaffold for hESC-RPE transplantation, which could be a new grafting material for tissue engineering RPE cells.

Cell Sources for Retinal Regeneration: Implication for Data Translation in Biomedicine of the Eye.

The main degenerative diseases of the retina include macular degeneration, proliferative vitreoretinopathy, retinitis pigmentosa, and glaucoma. Novel approaches for treating retinal diseases are based on cell replacement therapy using a variety of exogenous stem cells. An alternative and complementary approach is the potential use of retinal regeneration cell sources (RRCSs) containing retinal pigment epithelium, ciliary body, Müller glia, and retinal ciliary region. RRCSs in lower vertebrates in vivo and in mammals mostly in vitro are able to proliferate and exhibit gene expression and epigenetic characteristics typical for neural/retinal cell progenitors. Here, we review research on the factors controlling the RRCSs' properties, such as the cell microenvironment, growth factors, cytokines, hormones, etc., that determine the regenerative responses and alterations underlying the RRCS-associated pathologies. We also discuss how the current data on molecular features and regulatory mechanisms of RRCSs could be translated in retinal biomedicine with a special focus on (1) attempts to obtain retinal neurons de novo both in vivo and in vitro to replace damaged retinal cells; and (2) investigations of the key molecular networks stimulating regenerative responses and preventing RRCS-related pathologies.

GSK3 Is a Central Player in Retinal Degenerative Diseases but a Challenging Therapeutic Target.

Glycogen synthase kinase 3 (GSK3) is a key regulator of many cellular signaling processes and performs a wide range of biological functions in the nervous system. Due to its central role in numerous cellular processes involved in cell degeneration, a rising number of studies have highlighted the interest in developing therapeutics targeting GSK3 to treat neurodegenerative diseases. Although recent works strongly suggest that inhibiting GSK3 might also be a promising therapeutic approach for retinal degenerative diseases, its full potential is still under-evaluated. In this review, we summarize the literature on the role of GSK3 on the main cellular functions reported as deregulated during retinal degeneration, such as glucose homeostasis which is critical for photoreceptor survival, or oxidative stress, a major component of retinal degeneration. We also discuss the interest in targeting GSK3 for its beneficial effects on inflammation, for reducing neovascularization that occurs in some retinal dystrophies, or for cell-based therapy by enhancing Müller glia cell proliferation in diseased retina. Together, although GSK3 inhibitors hold promise as therapeutic agents, we highlight the complexity of targeting such a multitasked kinase and the need to increase our knowledge of the impact of reducing GSK3 activity on these multiple cellular pathways and biological processes.

Nerve Growth Factor Promotes Retinal Neurovascular Unit Repair: A Review.

Purpose: The purpose of this paper is to investigate how the imbalance of neurogenic factor (NGF) and its precursor (pro-NGF) mediates structural and functional impairment of retinal neurovascular unit (RNVU) that plays a role in retinal degenerative diseases.Methods: A literature search of electronic databases was performed.Results: The pro-apoptotic effect of pro-NGF and the pro-growth effect of NGF are essential for the pathological and physiological activities of RNVU. Studies show that NGF-based treatment of retinal degenerative diseases, including glaucoma, age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy, has achieved remarkable efficacy.Conclusions: RNVU plays a complex and multifaceted role in retinal degenerative diseases. The exploration of the differential signaling expression of proNGF-NGF homeostasis under physiological and pathological conditions, and the corresponding pathological processes induced by its regulation, has prompted us to focus on earlier retinal neuroprotective therapeutic strategies to prevent retinal degenerative diseases.

Contribution of Interleukin-17A to Retinal Degenerative Diseases.

Retinal degenerative diseases are a leading cause of vision loss and blindness throughout the world, characterized by chronic and progressive loss of neurons and/or myelin. One of the common features of retinal degenerative diseases and central neurodegenerative diseases is chronic neuroinflammation. Interleukin-17A (IL-17A) is the cytokine most closely related to disease in its family. Accumulating evidence suggests that IL-17A plays a key role in human retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and glaucoma. This review aims to provide an overview of the role of IL-17A participating in the pathogenesis of retinal degenerative diseases, which may open new avenues for potential therapeutic interventions.

Human primitive mesenchymal stem cell-derived retinal progenitor cells improved neuroprotection, neurogenesis, and vision in rd12 mouse model of retinitis pigmentosa.

BACKGROUND: Currently, there is no treatment for retinal degenerative diseases (RDD) such as retinitis pigmentosa (RP). Stem cell-based therapies could provide promising opportunities to repair the damaged retina and restore vision. Thus far, primarily adult mesenchymal stem cells (MSCs) have been investigated in preclinical and clinical studies, and the results have not been convincing. We applied a new approach in which primitive (p) MSC-derived retinal progenitor cells (RPCs) were examined to treat retinal degeneration in an rd12 mouse model of RP. METHODS: Well-characterized pMSCs and RPCs labeled with PKH26 were intravitreally injected into rd12 mice. The vision and retinal function of transplanted animals were analyzed using electroretinography. Animals were killed 4 and 8 weeks after cell transplantation for histological, immunological, molecular, and transcriptomic analyses of the retina. RESULTS: Transplanted RPCs significantly improved vision and retinal thickness as well as function in rd12 mice. pMSCs and RPCs homed to distinct retinal layers. pMSCs homed to the retinal pigment epithelium, and RPCs migrated to the neural layers of the retina, where they improved the thickness of the respective layers and expressed cell-specific markers. RPCs induced anti-inflammatory and neuroprotective responses as well as upregulated the expression of genes involved in neurogenesis. The transcriptomic analysis showed that RPCs promoted neurogenesis and functional recovery of the retina through inhibition of BMP and activation of JAK/STAT and MAPK signaling pathways. CONCLUSIONS: Our study demonstrated that RPCs countered inflammation, provided retinal protection, and promoted neurogenesis resulting in improved retinal structure and physiological function in rd12 mice.

New insights into the molecular mechanism of rhodopsin retinitis pigmentosa from the biochemical and functional characterization of G90V, Y102H and I307N mutations.

Mutations in the photoreceptor protein rhodopsin are known as one of the leading causes of retinal degeneration in humans. Two rhodopsin mutations, Y102H and I307N, obtained in chemically mutagenized mice, are currently the subject of increased interest as relevant models for studying the process of retinal degeneration in humans. Here, we report on the biochemical and functional characterization of the structural and functional alterations of these two rhodopsin mutants and we compare them with the G90V mutant previously analyzed, as a basis for a better understanding of in vivo studies. This mechanistic knowledge is fundamental to use it for developing novel therapeutic approaches for the treatment of inherited retinal degeneration in retinitis pigmentosa. We find that Y102H and I307N mutations affect the inactive-active equilibrium of the receptor. In this regard, the mutations reduce the stability of the inactive conformation but increase the stability of the active conformation. Furthermore, the initial rate of the functional activation of transducin, by the I307N mutant is reduced, but its kinetic profile shows an unusual increase with time suggesting a profound effect on the signal transduction process. This latter effect can be associated with a change in the flexibility of helix 7 and an indirect effect of the mutation on helix 8 and the C-terminal tail of rhodopsin, whose potential role in the functional activation of the receptor has been usually underestimated. In the case of the Y102H mutant, the observed changes can be associated with conformational alterations affecting the folding of the rhodopsin intradiscal domain, and its presumed involvement in the retinal binding process by the receptor.

Potential of Müller Glial Cells in Regeneration of Retina; Clinical and Molecular Approach.

Retinal degenerative diseases are a group of heterogeneous eye diseases that affect a significant percentage of the world's population, i.e., age-related macular degeneration (AMD), diabetic retinopathy, retinitis pigmentosa (RP), and glaucoma. Regenerative medicines look for novel therapies for severe injuries or chronic diseases, e.g., retina degeneration. Müller glia is the only retinal glia type with a common embryonic origin, with retinal neurons derived from the neural crest. Also, the lack of neurons in the retina does not automatically regenerate. Therefore, Müller glial cells, which make up about 5% of retinal cells, are a potent source for retinal regeneration. Following the retinal damage, Müller glial cells dedifferentiate and re-enter the cell cycle, producing multipotent progenitor cells. This feature leads to applying Müller glial cells in the regeneration of the retina. This study reviews this feature's molecular and clinical approaches, focusing on the critical signaling pathways, generation and transplantation methods, and clinical and sub-clinical challenges.

An audio-visual motor training improves audio spatial localization skills in individuals with scotomas due to retinal degenerative diseases.

Several studies have shown that impairments in a sensory modality can induce perceptual deficits in tasks involving the remaining senses. For example, people with retinal degenerative diseases like Macular Degeneration (MD) and with central scotoma show biased auditory localization abilities towards the visual field's scotoma area. This result indicates an auditory spatial reorganization of cross-modal processing in people with scotoma when the visual information is impaired. Recent works showed that multisensory training could be beneficial to improve spatial perception. In line with this idea, here we hypothesize that audio-visual and motor training could improve people's spatial skills with retinal degenerative diseases. In the present study, we tested this hypothesis by testing two groups of scotoma patients in an auditory and visual localization task before and after a training or rest performance. The training group was tested before and after multisensory training, while the control group performed the two tasks twice after 10 min of break. The training was done with a portable device positioned on the finger, providing spatially and temporally congruent audio and visual feedback during arm movement. Our findings show improved audio and visual localization for the training group and not for the control group. These results suggest that integrating multiple spatial sensory cues can improve the spatial perception of scotoma patients. This finding ignites further research and applications for people with central scotoma for whom rehabilitation is classically focused on training visual modality only.

Development-related mitochondrial properties of retinal pigment epithelium cells derived from hEROs.

AIM: To explore the temporal mitochondrial characteristics of retinal pigment epithelium (RPE) cells obtained from human embryonic stem cells (hESC)-derived retinal organoids (hEROs-RPE), to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration (AMD). METHODS: RPE cells were obtained from hEROs and from spontaneous differentiation (SD-RPE). The mitochondrial characteristics were analyzed every 20d from day 60 to 160. Mitochondrial quantity was measured by MitoTracker Green staining. Transmission electron microscopy (TEM) was adopted to assess the morphological features of the mitochondria, including their distribution, length, and cristae. Mitochondrial membrane potentials (MMPs) were determined by JC-1 staining and evaluated by flow cytometry, reactive oxygen species (ROS) levels were evaluated by flow cytometry, and adenosine triphosphate (ATP) levels were measured by a luminometer. Differences between two groups were analyzed by the independent-samples t-test, and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance was not assumed. RESULTS: hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE markers (Pax6, MITF, Bestrophin-1, RPE65, Cralbp). RPE features, including a cobblestone-like morphology with tight junctions (ZO-1), pigments and microvilli, were also observed in both hEROs-RPE and SD-RPE cells. The mitochondrial quantities of hEROs-RPE and SD-RPE cells both peaked at day 80. However, the cristae of hEROs-RPE mitochondria were less mature and abundant than those of SD-RPE mitochondria at day 80, with hEROs-RPE mitochondria becoming mature at day 100. Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period. However, hEROs-RPE mitochondria maintained a longer time to produce high levels of ATP (from day 120 to 140) than SD-RPE cells (only day 120). CONCLUSION: hEROs-RPE mitochondria develop more slowly and maintain a longer time to supply high-level energy than SD-RPE mitochondria. From the mitochondrial perspective, hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.