Retinal degeneration, characterized by the progressive loss of photoreceptors, retinal pigment epithelium cells, and/or ganglion cells, is a leading cause of vision impairment. These diseases are generally classified as inherited (e.g., retinitis pigmentosa, Stargardt disease) or acquired (e.g., age-related macular degeneration, diabetic retinopathy, glaucoma) ocular disorders that can lead to blindness. Available treatment options focus on managing symptoms or slowing disease progression and do not address the underlying causes of these diseases. However, recent advancements in regenerative medicine offer alternative solutions for repairing or protecting degenerated retinal tissue. Stem and progenitor cell therapies have shown great potential to differentiate into various retinal cell types and can be combined with gene editing, extracellular vesicles and exosomes, and bioactive molecules to modulate degenerative cellular pathways. Additionally, gene therapy and neuroprotective molecules play a crucial role in enhancing the efficacy of regenerative approaches. These innovative strategies hold the potential to halt the progression of retinal degenerative disorders, repair or replace damaged cells, and improve visual function, ultimately leading to a better quality of life for those affected.

Outer retinal degenerative diseases (RDDs) and injuries leading to photoreceptor (PR) loss are prevailing causes of blindness worldwide. While significant progress has been made in the manufacture of human pluripotent stem cell (hPSC)-derived PRs, robust production of pluripotent stem cell (PSC)-PRs from swine, a popular preclinical large animal model, would provide an avenue to collect conspecific functional and safety data to complement human xenograft studies. Toward this goal, we describe the highly efficient generation of PR-dominant porcine induced PSC (piPSC)-derived retinal organoids (ROs) using modifications of our established hPSC-RO differentiation protocol. Porcine iPSC-ROs were characterized using immunocytochemistry (ICC) and single-cell RNA sequencing (scRNA-seq), which revealed the presence and maturation of major neural retina cell types, including PRs and retinal ganglion cells, which possess molecular signatures akin to those found in hPSC-ROs. In late piPSC-ROs, a highly organized outer neuroepithelium was observed with rods and cones possessing outer segments and axon terminals expressing pre-synaptic markers adjacent to dendritic terminals of bipolar cells. The existence of piPSC lines and protocols that support reproducible, scalable production of female and male ROs will facilitate transplant studies in porcine models of retinal injury and RDDs unconfounded by immunological and evolutionary incompatibilities inherent to human xenografts.

Self-renewal capacity and potential to differentiate into almost any cell type of the human body makes pluripotent stem cells a valuable starting material for manufacturing of clinical grade cell therapies. Neurodegenerative diseases are characterized by gradual loss of structure or function of neurons, often leading to neuronal death. This results in gradual decline of cognitive, motor, and physiological functions due to the degeneration of the central nervous systems. Over the past two decades, comprehensive preclinical efficacy (proof-of-concept) and safety studies have led to the initiation of First-in-Human phase I-II clinical trials for a range of neurodegenerative diseases. In this review, we explore the fundamentals and challenges of neural-cell therapies derived from pluripotent stem cells for treating neurodegenerative diseases. Additionally, we highlight key preclinical investigations that paved the way for regulatory approvals of these trials. Furthermore, we provide an overview on progress and status of clinical trials done so far in treating neurodegenerative diseases such as spinal cord injury (SCI), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), as well as advances in retina diseases such as Stargardt disease (a.k.a fundus flavimaculatus), retinitis pigmentosa (RP) and age-related macular degeneration (AMD). These trials will pave the way for the development of new cell-based therapies targeting additional neurological conditions, including Alzheimer's disease and epilepsy.

Despite the crucial role of synaptic connections and neural activity in the development and organization of cortical circuits, the mechanisms underlying the formation of functional synaptic connections in the developing human cerebral cortex remain unclear. We investigated the development of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission using human cortical organoids (hCOs) derived from induced pluripotent stem cells. Two-photon Ca2⁺ imaging revealed an increase in the frequency and amplitude of spontaneous activity in hCOs on day 80 compared to day 50. Additionally, spontaneous neural activity in late-stage hCOs, but not in early-stage hCOs, was blocked by N-methyl-D-aspartate receptor (NMDAR) and AMPAR antagonists. However, transsynaptic circuit tracing with G-deleted rabies viral vectors indicated a similar number of synaptic connections in early- and late-stage hCOs. Notably, chemical labeling demonstrated a significant increase in AMPAR expression on the postsynaptic membrane and colocalization with NMDARs in late-stage hCOs. These results suggest that hCOs progressively organize excitatory synaptic transmission, concurrent with the transition from silent synapses lacking AMPARs to functional synapses containing NMDARs and AMPARs. This in vitro model of human cortical circuits derived from induced pluripotent stem cells reflects the developmental programs underlying physiological transitions, providing valuable insights into human corticogenesis and neurodevelopmental disorders.

Photoreceptor cell replacement therapy for retinal degenerative diseases is a promising approach. Presently, most protocols aimed at generating clinically safe and functional cells for retinal diseases face challenges such as low efficiency, poor reproducibility, and time-consuming and complex procedures. These could be due to the dependency on animal-derived components in cell culture media and substrates that support the cell differentiation process. Such conditions are poorly defined chemically, which could affect the robustness of the method and hinder clinical translation of cell therapy in retinal diseases. Here, we describe a simple protocol that is xenogen free and chemically defined to differentiate human embryonic stem cells to photoreceptor progenitors. Human recombinant extracellular matrix laminin 523 and 521 isoforms were used to mimic the inter-photoreceptor matrix niche environment to promote the retinal cell differentiation process. This was also accomplished by the unique combination of two cell differentiation media that recapitulates the retinal development signaling processes. In comparison to other protocols, our protocol does not require any mechanical dissection, which can be technically subjective and tedious. Our directed differentiation method generates photoreceptor progenitors that express ~17% cone-rod homeobox (CRX) transcript based on single-cell transcriptomic analyses by day 32. These day 32 photoreceptor progenitors can be cryopreserved and still maintain high cell viability after thawing for cell transplantation. This protocol can be easily reproduced and performed by researchers with basic cell culture experience, which is particularly important for retinal research progress and clinical cell manufacturing in a Good Manufacturing Practice facility.

Retinal degenerative diseases encompass a diverse range of eye conditions that result in blindness, many due to photoreceptor dysfunction and loss. Regrettably, current clinical treatments are frequently not overly effective. However, photoreceptor transplantation shows promise as a potential therapy for late-stage retinal degenerative diseases. This article will review the various donor cell sources for this transplantation, as well as the mechanisms and factors that impact donor cell integration and material transfer, donor cell maturation, and other auxiliary methods that can be combined with photoreceptor transplantation to treat these degenerative retinal diseases.

The inherent limitations of traditional treatments for Diabetic Retinopathy (DR) have spurred the development of various nanotechnologies, offering a safer and more efficient approach to managing the disease. Nanomedicine platforms present promising advancements in the diagnosis and treatment of DR by enhancing imaging capabilities, enabling targeted and controlled drug delivery. These innovations ultimately lead to more effective and personalized treatments with fewer side effects. This review highlights the progress, challenges, and opportunities in developing effective diagnostics and therapeutics for DR. Additionally, it explores innovative engineering techniques that leverage our growing understanding of nano-bio interactions to create more potent nanotherapeutics for patients.

Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.

Retinitis pigmentosa represents a leading cause of blindness in developed countries, yet effective treatments for the disease remain unestablished. Previous studies have demonstrated the potential of stem cell-derived retinal organoid (SC-RO) sheet transplantation to form host-graft synapses and to improve light responsiveness in animal models of retinal degeneration. However, the detailed microstructures of these de novo synapses and their functional contribution have not been well elucidated. This study aims to (1) elucidate the microstructures of the host-graft synapse, and (2) investigate the overall distribution and contribution of these synapses to host retinal light responses.

We identified host-graft synapses using a reporter system in mouse SC-RO and rd1 mice, a well-established model of end-stage retinal degeneration. Correlative array tomography was used to reveal the microstructure of host-graft synapses. Furthermore, we developed a semi-automated algorithm that robustly detects the host-graft photoreceptor synapses in the overall grafted area using the same reporter system in flat-mount retinas. We then integrated the spatial distribution of the host-graft synapses with light responses detected by multi-electrode array recording.

Correlative array tomography revealed that host-graft synapses recapitulate the developmental process of photoreceptor synapse formation involving horizontal cells first and then rod bipolar cells. By integrating the spatial distribution of host-graft synapse and multi-electrode array recording, we showed that the number of light-responsive host retinal ganglion cells is positively correlated with the local density of host-graft synapses.

De novo host-graft synapses recapitulate the developmental microstructure of the photoreceptor synapse, and their formation contributes to the light responsiveness after SC-RO transplantation.

The retinal fovea in human and nonhuman primates is essential for high acuity and color vision. Within the fovea lies specialized circuitry in which signals from a single cone photoreceptor are largely conveyed to one ON and one OFF type midget bipolar cell (MBC), which in turn connect to a single ON or OFF midget ganglion cell (MGC), respectively. Restoring foveal vision requires not only photoreceptor replacement but also appropriate reconnection with surviving ON and OFF MBCs and MGCs. However, our current understanding of the effects of cone loss on the remaining foveal midget pathway is limited. We thus used serial block-face electron microscopy to determine the degree of plasticity and potential remodeling of this pathway in adult Macaca fascicularis several months after acute photoreceptor loss upon photocoagulation. We reconstructed MBC structure and connectivity within and adjacent to the region of cone loss. We found that MBC dendrites within the scotoma retracted and failed to reach surviving cones to form new connections. However, both surviving cones and ON and OFF MBC dendrites at the scotoma border exhibited remodeling, suggesting that these neurons can demonstrate plasticity and rewiring at maturity. At six months postlesion, disconnected OFF MBCs clearly lost output ribbon synapses with their postsynaptic partners, whereas the majority of ON MBCs maintained their axonal ribbon numbers, suggesting differential timing or extent in ON and OFF midget circuit remodeling after cone loss. Our findings raise rewiring considerations for cell replacement approaches in the restoration of foveal vision.

Retinal degeneration (RD) is a leading cause of blindness worldwide and includes conditions such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD). These diseases result in the permanent loss of vision due to the progressive and irreversible degeneration of retinal cells, including photoreceptors (PR) and the retinal pigment epithelium (RPE). The adult human retina has limited abilities to regenerate and repair itself, making it challenging to achieve complete self-replenishment and functional repair of retinal cells. Currently, there is no effective clinical treatment for RD. Stem cell therapy, which involves transplanting exogenous stem cells such as retinal progenitor cells (RPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs), or activating endogenous stem cells like Müller Glia (MG) cells, holds great promise for regenerating and repairing retinal cells in the treatment of RD. Several preclinical and clinical studies have shown the potential of stem cell-based therapies for RD. However, the clinical translation of these therapies for the reconstruction of substantial vision still faces significant challenges. This review provides a comprehensive overview of stem/progenitor cell-based therapy strategies for RD, summarizes recent advances in preclinical studies and clinical trials, and highlights the major challenges in using stem/progenitor cell-based therapies for RD.

Since the discovery of induced pluripotent stem cell (iPSC) technology, there have been many attempts to create cellular models of inherited retinal diseases (IRDs) for investigation of pathogenic processes to facilitate target discovery and validation activities. Consistency remains key in determining the utility of these findings. Despite the importance of consistency, quality control metrics are still not widely used. In this review, a toolkit for harnessing iPSC technology to generate photoreceptor, retinal pigment epithelial cell, and organoid disease models is provided. Considerations while developing iPSC-derived IRD models such as iPSC origin, reprogramming methods, quality control metrics, control strategies, and differentiation protocols are discussed. Various iPSC IRD models are dissected and the scientific hurdles of iPSC-based disease modeling are discussed to provide an overview of current methods and future directions in this field.

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, offer groundbreaking therapeutic potential for degenerative diseases and cellular repair. Despite their significance, a comprehensive bibliometric analysis in this field, particularly in relation to age-related macular degeneration (AMD), is yet to be conducted. This study aims to map the foundational and emerging areas in stem cell and AMD research through bibliometric analysis.

This study analyzed articles and reviews on stem cells and AMD from 2000 to 2022, sourced from the Web of Science Core Collection. We used VOSviewer and CiteSpace for analysis and visualization of data pertaining to countries, institutions, authors, journals, references and key words. Statistical analyses were conducted using R language and Microsoft Excel 365.

In total, 539 publications were included, indicating an increase in global literature on stem cells and AMD from 2000 to 2022. The USA was the leading contributor, with 239 papers and the highest H-index, also the USA had the highest average citation rate per article (59.82). Notably, 50% of the top 10 institutions were from the USA, with the University of California system being the most productive. Key authors included Masayo Takahashi, Michiko Mandai, Dennis Clegg, Pete J. Coffey, Boris Stanzel, and Budd A. Tucker. Investigative Ophthalmology & Visual Science published the majority of relevant papers (n = 27). Key words like "clinical trial," "stem cell therapy," "retinal organoid," and "retinal progenitor cells" were predominant.

Research on stem cells and AMD has grown significantly, highlighting the need for increased global cooperation. Current research prioritizes the relationship between "ipsc," "induced pluripotent stem cell," "cell culture," and "human embryonic stem cell." As stem cell culture and safety have advanced, focus has shifted to prognosis and complications post-transplantation, signifying the movement of stem cell research from labs to clinical settings.

Blindness poses a growing global challenge, with approximately 26% of cases attributed to degenerative retinal diseases. While gene therapy, optogenetic tools, photosensitive switches, and retinal prostheses offer hope for vision restoration, these high-cost therapies will benefit few patients. Understanding retinal diseases is therefore key to advance effective treatments, requiring in vitro models replicating pathology and allowing quantitative assessments for drug discovery. Pluripotent stem cells (PSCs) provide a unique solution given their limitless supply and ability to differentiate into light-responsive retinal tissues encompassing all cell types. This review focuses on the history and current state of photoreceptor and retinal pigment epithelium (RPE) cell generation from PSCs. We explore the applications of this technology in disease modelling, experimental therapy testing, biomarker identification, and toxicity studies. We consider challenges in scalability, standardisation, and reproducibility, and stress the importance of incorporating vasculature and immune cells into retinal organoids. We advocate for high-throughput automation in data acquisition and analyses and underscore the value of advanced micro-physiological systems that fully capture the interactions between the neural retina, RPE, and choriocapillaris.

Adult stem cells, present in various parts of the human body, are undifferentiated cells that can proliferate and differentiate to replace dying cells within tissues. Stem cells have specifically been identified in the cornea, trabecular meshwork, crystalline lens, iris, ciliary body, retina, choroid, sclera, conjunctiva, eyelid, lacrimal gland, and orbital fat. The identification of ocular stem cells broadens the potential therapeutic strategies for untreatable eye diseases. Currently, stem cell transplantation for corneal and conjunctival diseases remains the most common stem cell-based therapy in ocular clinical management. Lens epithelial stem cells have been applied in the treatment of paediatric cataracts. Several early-phase clinical trials for corneal and retinal regeneration using ocular stem cells are also underway. Extensive preclinical studies using ocular stem cells have been conducted, showing encouraging outcomes. Ocular stem cells currently demonstrate great promise in potential treatments of eye diseases. In this review, we focus on the identification, characterisation, and therapeutic application of adult stem cells in the eye.

Pluripotent stem cell-based therapy for retinal degenerative diseases is a promising approach to restoring visual function. A clinical study using retinal organoid (RO) sheets was recently conducted in patients with retinitis pigmentosa. However, the graft preparation currently requires advanced skills to identify and excise suitable segments from the transplantable area of the limited number of suitable ROs. This remains a challenge for consistent clinical implementations. Herein, we enabled the enrichment of wild-type (non-reporter) retinal progenitor cells (RPCs) from dissociated ROs using a label-free ghost cytometry (LF-GC)-based sorting system, where a machine-based classifier was trained in advance with another RPC reporter line. The sorted cells reproducibly formed retinal spheroids large enough for transplantation and developed mature photoreceptors in the retinal degeneration rats. This method of enriching early RPCs with no specific surface antigens and without any reporters or chemical labeling is promising for robust preparation of graft tissues during cell-based therapy.

Titanium (Ti) and titanium alloy are the most common metal materials in clinical orthopedic surgery. However, in the initial stage of surgery and implantation, the production of excessive reactive oxygen species (ROS) can induce oxidative stress (OS) microenvironment. OS will further inhibit the growth of new bone, resulting in surgical failure. In this study, based on the fact that nanoscale manganese dioxide (MnO2) can show H2O2-like enzyme activity, a MnO2 nanocoating was prepared on mciro-nano structured surface of Ti substrate via a two-step method of alkaline thermal and hydrothermal treatment. The results of scanning electron microscopy (SEM), X-ray diffractometer (XRD) and X-ray photoelectron spectroscopy (XPS) showed that the nano-MnO2 coating was successfully fabricated on the surface of Ti substrate. The results of measurement of H2O2, dissolved O2 and intracellular ROS in vitro showed that the treated Ti substrate could efficiently eliminate H2O2 and reduce ROS. Furthermore, the modified Ti substrate could promote the early adhesion, proliferation and osteogenic differentiation of MSCs, which was demonstrated by experimental results of cell morphology, cell viability, alkaline phosphatase, collagen, and mineralization deposition. The results of quantitative real-time polymerase chain reaction (qRT-PCR) of MSCs adhered the modified Ti substrate showed that the expression of genes related to osteogenic differentiation significantly increased. More importantly, the modified Ti implant could eliminate ROS at the injury site, reduce OS and promote the regeneration of bone tissue, which was demonstrated via hematoxylin/eosin, Masson's trichrome and immunohistochemical staining. In conclusion, the modified Ti implant presented here had the effect of reducing OS and promoting osseointegration. Relevant research ideas and results provide new methods for the research and development of functional implants, which have potential application value in the field of orthopedics.

Retinal diseases are leading causes of blindness globally. Developing new drugs is of great significance for preventing vision loss. Current drug discovery relies mainly on two-dimensional in vitro models and animal models, but translation to human efficacy and safety is biased. In recent years, the emergence of retinal organoid technology platforms, utilizing three-dimensional microenvironments to better mimic retinal structure and function, has provided new platforms for exploring pathogenic mechanisms and drug screening. This review summarizes the latest advances in retinal organoid technology, emphasizing its application advantages in high-throughput drug screening, efficacy and toxicity evaluation, and translational medicine research. The review also prospects the combination of emerging technologies such as organ-on-a-chip, 3D bioprinting, single cell sequencing, gene editing with retinal organoid technology, which is expected to further optimize retinal organoid models and advance the diagnosis and treatment of retinal diseases.

NRL is an influential transcription factor and central to animal modeling in ophthalmology. Disrupting NRL abrogates rod development and produces an excess of S-cones (also known as "UV cones" or "short-wavelength-sensitive1 [SWS1] cones"). Strikingly, mutations in zebrafish tbx2b produce the exact opposite phenotypes (excess rods and loss of SWS1 cones). We sought to define what genetic relationship exists, if any, between these transcription factors. We also infer whether these two phenotypes (altered rod abundance and altered SWS1 cone abundance) are independent versus inter-related.

Zebrafish mutants were bred to disrupt nrl and tbx2b in concert. Rods and SWS1 cones were quantified and characterized at ultrastructural and transcriptional levels.

Considering single mutant zebrafish, we confirmed previously established phenotypes and noted that the number of rods lost in nrl-/- mutants is reflected by a concomitant increase in SWS1 cone abundance. The tbx2b-/- mutants present the opposite phenotype(s) but exhibit a similar trade-off in cell abundances, with lots of rods and a concomitant decrease in SWS1 cones. Double mutant nrl-/-;tbx2b-/- zebrafish recapitulate the nrl-/- mutant phenotype(s).

The tbx2b is thought to be required for producing SWS1 cones in zebrafish, but this can be over-ridden when nrl is absent. Regarding the altered cell abundances observed in either tbx2b-/- or nrl-/- mutants, the alterations in rod and SWS1 cones appear to not be two separate phenotypes but are instead a single intertwined outcome. The tbx2b and nrl are in an epistatic relationship, with nrl phenotypes dominating, implying that tbx2b is upstream of nrl in photoreceptor cell fate determination.

Retinal degeneration diseases affect millions of people worldwide but are among the most difficult eye diseases to cure. Studying the mechanisms and developing new therapies for these blinding diseases requires researchers to have access to many retinal cells. In recent years there has been substantial advances in the field of biotechnology in generating retinal cells and even tissues in vitro, either through programmed sequential stem cell differentiation or direct somatic cell lineage reprogramming. The resemblance of these in vitro-generated retinal cells to native cells has been increasingly utilized by researchers. With the help of these in vitro retinal models, we now have a better understanding of human retinas and retinal diseases. Furthermore, these in vitro-generated retinal cells can be used as donor cells which solves a major hurdle in the development of cell replacement therapy for retinal degeneration diseases, while providing a promising option for patients suffering from these diseases. In this review, we summarize the development of pluripotent stem cell-to-retinal cell differentiation methods, the recent advances in generating retinal cells through direct somatic cell reprogramming, and the translational applications of retinal cells generated in vitro. Finally, we discuss the limitations of the current protocols and possible future directions for improvement.

Primary cilia are specialized organelles on the surface of almost all cells in vertebrate tissues and are primarily involved in the detection of extracellular stimuli. In retinal photoreceptors, cilia are uniquely modified to form outer segments containing components required for the detection of light in stacks of membrane discs. Not surprisingly, vision impairment is a frequent phenotype associated with ciliopathies, a heterogeneous class of conditions caused by mutations in proteins required for formation, maintenance and/or function of primary cilia. Traditionally, immortalized cell lines and model organisms have been used to provide insights into the biology of ciliopathies. The advent of methods for reprogramming human somatic cells into pluripotent stem cells has enabled the generation of in vitro disease models directly from patients suffering from ciliopathies. Such models help us in investigating pathological mechanisms specific to human physiology and in developing novel therapeutic approaches. In this article, we review current protocols to differentiate human pluripotent stem cells into retinal cell types, and discuss how these cellular and/or organoid models can be utilized to interrogate pathobiology of ciliopathies affecting the retina and for testing prospective treatments.

The retina is a part of the brain that sits at the back of the eye, looking out onto the world. The first neurons of the retina are the rod and cone photoreceptors, which convert changes in photon flux into electrical signals that are the basis of vision. Rods and cones are frequent targets of heritable neurodegenerative diseases that cause visual impairment, including blindness, in millions of people worldwide. This review summarizes the diverse genetic causes of inherited retinal degenerations (IRDs) and their convergence onto common pathogenic mechanisms of vision loss. Currently, there are few effective treatments for IRDs, but recent advances in disparate areas of biology and technology (e.g., genome editing, viral engineering, 3D organoids, optogenetics, semiconductor arrays) discussed here enable promising efforts to preserve and restore vision in IRD patients with implications for neurodegeneration in less approachable brain areas.

Diseases leading to retinal cell loss can cause severe visual impairment and blindness. The lack of effective therapies to address retinal cell loss and the absence of intrinsic regeneration in the human retina leads to an irreversible pathological condition. Progress in recent years in the generation of human three-dimensional retinal organoids from pluripotent stem cells makes it possible to recreate the cytoarchitecture and associated cell-cell interactions of the human retina in remarkable detail. These human three-dimensional retinal organoid systems made of distinct retinal cell types and possessing contextual physiological responses allow the study of human retina development and retinal disease pathology in a way animal model and two-dimensional cell cultures were unable to achieve. We describe the derivation of retinal organoids from human pluripotent stem cells and their application for modeling retinal disease pathologies, while outlining the opportunities and challenges for its application in academia and industry.

Stem cell therapies can potentially treat various retinal degenerative diseases, including age-related macular degeneration (AMD) and inherited retinal diseases like retinitis pigmentosa. For these diseases, transplanted cells may include stem cell-derived retinal pigmented epithelial (RPE) cells, photoreceptors, or a combination of both. Although stem cell-derived RPE cells have progressed to human clinical trials, therapies using photoreceptors and other retinal cell types are lagging. In this review, we discuss the potential use of human pluripotent stem cell (hPSC)-derived photoreceptors for the treatment of retinal degeneration and highlight the progress and challenges for their efficient production and clinical application in regenerative medicine.

The human eye plays a critical role in vision perception, but various retinal degenerative diseases such as retinitis pigmentosa (RP), glaucoma, and age-related macular degeneration (AMD) can lead to vision loss or blindness. Although progress has been made in understanding retinal development and in clinical research, current treatments remain inadequate for curing or reversing these degenerative conditions. Animal models have limited relevance to humans, and obtaining human eye tissue samples is challenging due to ethical and legal considerations. Consequently, researchers have turned to stem cell-based approaches, specifically induced pluripotent stem cells (iPSCs), to generate distinct retinal cell populations and develop cell replacement therapies. iPSCs offer a novel platform for studying the key stages of human retinogenesis and disease-specific mechanisms. Stem cell technology has facilitated the production of diverse retinal cell types, including retinal ganglion cells (RGCs) and photoreceptors, and the development of retinal organoids has emerged as a valuable in vitro tool for investigating retinal neuron differentiation and modeling retinal diseases. This review focuses on the protocols, culture conditions, and techniques employed in differentiating retinal neurons from iPSCs. Furthermore, it emphasizes the significance of molecular and functional validation of the differentiated cells.

During brain and spinal cord development, GABA and glycine, the inhibitory neurotransmitters, cause depolarization instead of hyperpolarization in adults. Since glycine and GABAA receptors (GABAARs) are chloride (Cl-) ion channel receptor, the conversion of GABA/glycine actions during development is influenced by changes in the transmembrane Cl- gradient, which is regulated by Cl- transporters, NKCC1 (absorption) and KCC2 (expulsion). In immature neurons, inhibitory neurotransmitters are released in a non-vesicular/non-synaptic manner, transitioning to vesicular/synaptic release as the neuron matures. In other word, in immature neurons, neurotransmitters generally act tonically. Thus, the glycine/GABA system is a developmentally multimodal system that is required for neurogenesis, differentiation, migration, and synaptogenesis. The endogenous agonists for these receptors are not fully understood, we address taurine. In this review, we will discuss about the properties and function of taurine during development of neocortex. Taurine cannot be synthesized by fetuses or neonates, and is transferred from maternal blood through the placenta or maternal milk ingestion. In developing neocortex, taurine level is higher than GABA level, and taurine tonically activates GABAARs to control radial migration as a stop signal. In the marginal zone (MZ) of the developing neocortex, endogenous taurine modulates the spread of excitatory synaptic transmission, activating glycine receptors (GlyRs) as an endogenous agonist. Thus, taurine affects information processing and crucial developmental processes such as axonal growth, cell migration, and lamination in the developing cerebral cortex. Additionally, we also refer to the possible mechanism of taurine-regulating Cl- homeostasis. External taurine is uptake by taurine transporter (TauT) and regulates NKCC1 and KCC2 mediated by intracellular signaling pathway, with-no-lysine kinase 1 (WNK1) and its subsequent kinases STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Through the regulation of NKCC1 and KCC2, mediated by the WNK-SPAK/OSR1 signaling pathway, taurine plays a role in maintaining Cl- homeostasis during normal brain development.

Prior to use, newly generated induced pluripotent stem cells (iPSC) should be thoroughly validated. While excellent validation and release testing assays designed to evaluate potency, genetic integrity, and sterility exist, they do not have the ability to predict cell type-specific differentiation capacity. Selection of iPSC lines that have limited capacity to produce high-quality transplantable cells, places significant strain on valuable clinical manufacturing resources. The purpose of this study was to determine the degree and root cause of variability in retinal differentiation capacity between cGMP-derived patient iPSC lines. In turn, our goal was to develop a release testing assay that could be used to augment the widely used ScoreCard panel. IPSCs were generated from 15 patients (14-76 years old), differentiated into retinal organoids, and scored based on their retinal differentiation capacity. Despite significant differences in retinal differentiation propensity, RNA-sequencing revealed remarkable similarity between patient-derived iPSC lines prior to differentiation. At 7 days of differentiation, significant differences in gene expression could be detected. Ingenuity pathway analysis revealed perturbations in pathways associated with pluripotency and early cell fate commitment. For example, good and poor producers had noticeably different expressions of OCT4 and SOX2 effector genes. QPCR assays targeting genes identified via RNA sequencing were developed and validated in a masked fashion using iPSCs from 8 independent patients. A subset of 14 genes, which include the retinal cell fate markers RAX, LHX2, VSX2, and SIX6 (all elevated in the good producers), were found to be predictive of retinal differentiation propensity.

The intricate neural circuit of retina extracts salient features of the natural world and forms bioelectric impulse as the origin of vision. The early development of retina is a highly complex and coordinated process in morphogenesis and neurogenesis. Increasing evidence indicates that stem cells derived human retinal organoids (hROs) in vitro faithfully recapitulates the embryonic developmental process of human retina no matter in the transcriptome, cellular biology and histomorphology. The emergence of hROs greatly deepens on the understanding of early development of human retina. Here, we reviewed the events of early retinal development both in animal embryos and hROs studies, which mainly comprises the formation of optic vesicle and optic cup shape, differentiation of retinal ganglion cells (RGCs), photoreceptor cells (PRs) and its supportive retinal pigment epithelium cells (RPE). We also discussed the classic and frontier molecular pathways up to date to decipher the underlying mechanisms of early development of human retina and hROs. Finally, we summarized the application prospect, challenges and cutting-edge techniques of hROs for uncovering the principles and mechanisms of retinal development and related developmental disorder. hROs is a priori selection for studying human retinal development and function and may be a fundamental tool for unlocking the unknown insight into retinal development and disease.

Retinal diseases are a rising concern as major causes of blindness in an aging society; therapeutic options are limited, and the precise pathogenesis of these diseases remains largely unknown. Intraocular drug delivery and nanomedicines offering targeted, sustained, and controllable delivery are the most challenging and popular topics in ocular drug development and toxicological evaluation. Retinal organoids (ROs) and organoid-on-a-chip (ROoC) are both emerging as promising in-vitro models to faithfully recapitulate human eyes for retinal research in the replacement of experimental animals and primary cells. In this study, we review the generation and application of ROs resembling the human retina in cell subtypes and laminated structures and introduce the emerging engineered ROoC as a technological opportunity to address critical issues. On-chip vascularization, perfusion, and close inter-tissue interactions recreate physiological environments in vitro, whilst integrating with biosensors facilitates real-time analysis and monitoring during organogenesis of the retina representing engineering efforts in ROoC models. We also emphasize that ROs and ROoCs hold the potential for applications in modeling intraocular drug delivery in vitro and developing next-generation retinal drug delivery strategies.

Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their spontaneous neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs containing excitatory and inhibitory neurons showed mature neural activity with an unsynchronized pattern. The CAs represent a powerful in vitro model for studying excitatory and inhibitory neuron interactions, cortical dynamics, and their dysfunction. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.

Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors. This chemically defined and xenogen-free method enables reproducible production of photoreceptor progenitors within 32 days. We observed that the transplantation into rd10 mice were able to protect the host photoreceptor outer nuclear layer (ONL) up to 2 weeks post transplantation as measured by full-field electroretinogram. At 4 weeks post transplantation, the engrafted cells were found to survive, mature, and associate with the host's rod bipolar cells. Visual behavioral assessment using the water maze swimming test demonstrated visual improvement in the cell-transplanted rodents. At 20 weeks post transplantation, the maturing engrafted cells were able to replace the loss of host ONL by extensive association with host bipolar cells and synapses. Post-transplanted rabbit model also provided congruent evidence for synaptic connectivity with the degenerated host retina. The results may pave the way for the development of stem cell-based therapeutics for retina degeneration.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

We report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from mouse native retinae and induced pluripotent stem cell (iPSC)-derived retinal organoids at four matched stages of development spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, pseudo-time and RNA velocity analyses, to show that iPSC-derived 3D retinal organoids broadly recapitulate the native developmental trajectories. However, we observe relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in retinal organoids. We demonstrate that genes causing human retinopathies are enriched in cell-type specifying genes and identify a subset of disease-causing genes with expression profiles that are highly conserved between human retinae and murine retinal organoids. This study provides a resource to the community that will be useful to assess and further improve protocols for ex vivo recapitulation and study of retinal development.

Background: Glaucoma, a neurodegenerative disease of the retina, is the leading cause of irreversible blindness. Stem cells have therapeutic potential for glaucoma. However, few bibliometric studies have been published in this field. Concerning a visual map, this article aims to characterize the research context, cooperation relationship, hotspots, and trends concerning the application of stem cells in glaucoma research. Methods: Publications focusing on stem cell research and glaucoma were retrieved from the Web of Science Core Collection. VOSviewer, CiteSpace, Microsoft Excel, and Scimago Graphica were used to map the contributions of countries or regions, authors, organizations, and journals. Journal Impact Factor data were obtained from the Web of Science Core Collection. We analyzed the tendencies, hotspots, and knowledge networks using VOSviewer, and CiteSpace. Results: We analyzed 518 articles published from 1999 through 2022. In the first decade, the number of articles in this field increased slowly, and there was a marked acceleration in publication frequency after 2010. The United States, China, and England were the main contributors. Yiqin Du was the most prolific author, and among the top 10 prolific writers, Keith R. Martin's work was cited most frequently. Investigative Ophthalmology and Visual Science, Experimental Eye Research, and Cornea published the most articles in this domain. The three most commonly co-cited journals were Investigative Ophthalmology and Visual Science, Experimental Eye Research, and Proceedings of the National Academy of Sciences of the United States of America. The Central South University, the University of Pittsburgh, and the National Institutes of Health National Eye Institute were highly prolific institutions in this research area. Our keywords analysis with VOSviewer suggested directions of future research and yielded the following recent key themes, extracellular vesicles, exosomes, mitochondria, growth factors, oxidative stress, and ocular diseases. Four co-cited references had a citation burst duration until 2022. Conclusion: With improvements in overall quality of life and demographic transitions toward population aging, research and clinical focus on eye care has increased, with glaucoma as a key area of emphasis. This study added to our understanding of the global landscape and Frontier hotspots in this field.

Characterizing cell identity in complex tissues such as the human retina is essential for studying its development and disease. While retinal organoids derived from pluripotent stem cells have been widely used to model development and disease of the human retina, there is a lack of studies that have systematically evaluated the molecular and cellular fidelity of the organoids derived from various culture protocols in recapitulating their in vivo counterpart. To this end, we performed an extensive meta-atlas characterization of cellular identities of the human eye, covering a wide range of developmental stages. The resulting map uncovered previously unknown biomarkers of major retinal cell types and those associated with cell-type-specific maturation. Using our retinal-cell-identity map from the fetal and adult tissues, we systematically assessed the fidelity of the retinal organoids in mimicking the human eye, enabling us to comprehensively benchmark the current protocols for retinal organoid generation.

Zika virus (ZIKV) causes microcephaly and congenital eye disease. The cellular and molecular basis of congenital ZIKV infection are not well understood. Here, we utilized a biologically relevant cell-based system of human fetal retinal pigment epithelial cells (FRPEs), hiPSC-derived retinal stem cells (iRSCs), and retinal organoids to investigate ZIKV-mediated ocular cell injury processes. Our data show that FRPEs were highly susceptible to ZIKV infection exhibiting increased apoptosis, whereas iRSCs showed reduced susceptibility. Detailed transcriptomics and proteomics analyses of infected FRPEs were performed. Nucleoside analogue drug treatment inhibited ZIKV replication. Retinal organoids were susceptible to ZIKV infection. The Asian genotype ZIKV exhibited higher infectivity, induced profound inflammatory response, and dysregulated transcription factors involved in retinal organoid differentiation. Collectively, our study shows that ZIKV affects ocular cells at different developmental stages resulting in cellular injury and death, further providing molecular insight into the pathogenesis of congenital eye disease.

Retinitis pigmentosa (RP) causes blindness in 1 out of 3000-4000 individuals worldwide. Understanding the disease mechanism underlying the death of photoreceptors in RP patient is crucial for the discovery and development of therapies to prevent and stop the progression of retinal degeneration. Despite having provided valuable insight into RP pathology, several shortcomings of animal models warrant the need for a better modeling system. This review discusses the current use of patient-derived induced pluripotent stem cells (iPSCs) to model RP and its advantages over animal models. Further improvement to enhance the representativeness of iPSC RP models is also discussed.

Photoreceptors (PRs), as the most abundant and light-sensing cells of the neuroretina, are responsible for converting light into electrical signals that can be interpreted by the brain. PR degeneration, including morphological and functional impairment of these cells, causes significant diminution of the retina's ability to detect light, with consequent loss of vision. Recent findings in ocular regenerative medicine have opened promising avenues to apply neuroprotective therapy, gene therapy, cell replacement therapy, and visual prostheses to the challenge of restoring vision. However, successful visual restoration in the clinical setting requires application of these therapeutic approaches at the appropriate stage of the retinal degeneration. In this review, firstly, we discuss the mechanisms of PR degeneration by focusing on the molecular mechanisms underlying cell death. Subsequently, innovations, recent developments, and promising treatments based on the stage of disorder progression are further explored. Then, the challenges to be addressed before implementation of these therapies in clinical practice are considered. Finally, potential solutions to overcome the current limitations of this growing research area are suggested. Overall, the majority of current treatment modalities are still at an early stage of development and require extensive additional studies, both pre-clinical and clinical, before full restoration of visual function in PR degeneration diseases can be realized.

Stem cell therapy is a current world-wide topic in medical science. Various therapies have been approved based on their effectiveness and put into practical use. In Japan, research and development-related stem cell therapy, generally referred to as regenerative medicine, has been led by the government. The national scheme started in 2002, and support for the transition to clinical trials has been accelerating since 2011. Of the initial 18 projects that were accepted in the budget for preclinical research, 15 projects have begun clinical trials so far. These include the transplantation of retinal, cardiac, and dopamine-producing cells differentiated from human induced pluripotent stem (iPS) cells and hepatocyte-like cells differentiated from human embryonic stem (ES) cells. The distinctive feature of the stem cell research in Japan is the use of iPS cells. A national framework was also been set-up to attain the final goal: health insurance coverage. Now, insurance covers cell transplantation therapies for the repair and recovery of damaged skin, articular cartilage, and stroke as well as therapies introduced from abroad, such as allogeneic mesenchymal stem cells for graft-versus-host disease and chimeric antigen receptor-T (CAR-T) cell therapy. To prepare this review, original information was sought from Japanese authentic websites, which are reliable but a little hard to access due to the fact of multiple less-organized databases and the language barrier. Then, each fact was corroborated by citing its English version or publication in international journals as much as possible. This review provides a summary of progress over the past decade under the national program and a state-of-the-art factual view of research activities, government policy, and regulation in Japan for the realization of stem cell therapy.

Retinal organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that mimic the retina's spatial and temporal differentiation, making them useful as in vitro retinal development models. Retinal organoids can be assembled with brain organoids, the 3D self-assembled aggregates derived from hPSCs containing different cell types and cytoarchitectures that resemble the human embryonic brain. Recent studies have shown the development of optic cups in brain organoids. The cellular components of a developing optic vesicle-containing organoids include primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. The importance of retinal organoids in ocular diseases such as age-related macular degeneration, Stargardt disease, retinitis pigmentosa, and diabetic retinopathy are described in this review. This review highlights current developments in retinal organoid techniques, and their applications in ocular conditions such as disease modeling, gene therapy, drug screening and development. In addition, recent advancements in utilizing extracellular vesicles secreted by retinal organoids for ocular disease treatments are summarized.