ABSTRACT
Here, we present a protocol for isolating and culturing mouse photoreceptors in a minimal, chemically defined medium free from serum. We describe steps for retina dissection, enzymatic dissociation, photoreceptor enrichment, cell culture, extracellular vesicles (EVs) enrichment, and EV ultrastructural analysis. This protocol, which has been verified for cultured cells derived from multiple murine strains, allows for the study of several aspects of photoreceptor biology, including EV isolation and nanotube formation. For complete details on the use and execution of this protocol, please refer to Kalargyrou et al. (2021).1.
Subject(s)
Extracellular Vesicles , Retina , Animals , Mice , Cell Culture Techniques , DissectionABSTRACT
Combination therapies have become increasingly researched and used in the treatment and management of complex diseases due to their ability to increase the chances for better efficacy and decreased toxicity. To evaluate drug combinations in drug development, pharmacokinetic and pharmacodynamic interactions between drugs in combination can be quantified using mathematical models; however, it can be difficult to deduce which models to use and how to use them to aid in clinical trial simulations to simulate the effect of a drug combination. This review paper aims to provide an overview of the various methods used to evaluate combination drug interaction for use in clinical trial development and a practical guideline on how combination modeling can be used in the settings of clinical trials.
Subject(s)
Drug Development , Models, Theoretical , Humans , Drug Interactions , Drug Combinations , Drug Design , Drug Therapy, CombinationABSTRACT
The retina encompasses a network of neurons, glia and epithelial and vascular endothelia cells, all coordinating visual function. Traditionally, molecular information exchange in this tissue was thought to be orchestrated by synapses and gap junctions. Recent findings have revealed that many cell types are able to package and share molecular information via extracellular vesicles (EVs) and the technological advancements in visualisation and tracking of these delicate nanostructures has shown that the role of EVs in cell communication is pleiotropic. EVs are released under physiological conditions by many cells but they are also released during various disease stages, potentially reflecting the health status of the cells in their cargo. Little is known about the physiological role of EV release in the retina. However, administration of exogenous EVs in vivo after injury suggest a neurotrophic role, whilst photoreceptor transplantation in early stages of retina degeneration, EVs may facilitate interactions between photoreceptors and Müller glia cells. In this review, we consider some of the proposed roles for EVs in retinal physiology and discuss current evidence regarding their potential impact on ocular therapies via gene or cell replacement strategies and direct intraocular administration in the diseased eye.
ABSTRACT
The developing retina undergoes dynamic organizational changes involving significant intra-retinal motility of the encompassing cells. Here, we present a protocol for tracking retinal cell motility in live explanted mouse retinae. Although originally applied to rod and cone photoreceptors, this strategy is applicable to any fluorescently labeled cell in mouse retinae and other similar experimental retinal models. Careful tissue handling is critical for the successful acquisition of high-quality live imaging data. Further instructions for semi-automated in silico data handling are provided. For complete details on the use and execution of this protocol, please refer to Aghaizu et al. (2021).
Subject(s)
Cell Movement/physiology , Cell Tracking/methods , Retina , Retinal Cone Photoreceptor Cells , Retinal Rod Photoreceptor Cells , Animals , Female , Luminescent Proteins , Male , Mice , Mice, Transgenic , Microscopy, Fluorescence , Retina/cytology , Retina/diagnostic imaging , Retinal Cone Photoreceptor Cells/cytology , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/physiology , Time-Lapse ImagingABSTRACT
Neuronal communication is typically mediated via synapses and gap junctions. New forms of intercellular communication, including nanotubes (NTs) and extracellular vesicles (EVs), have been described for non-neuronal cells, but their role in neuronal communication is not known. Recently, transfer of cytoplasmic material between donor and host neurons ("material transfer") was shown to occur after photoreceptor transplantation. The cellular mechanism(s) underlying this surprising finding are unknown. Here, using transplantation, primary neuronal cultures and the generation of chimeric retinae, we show for the first time that mammalian photoreceptor neurons can form open-end NT-like processes. These processes permit the transfer of cytoplasmic and membrane-bound molecules in culture and after transplantation and can mediate gain-of-function in the acceptor cells. Rarely, organelles were also observed to transfer. Strikingly, use of chimeric retinae revealed that material transfer can occur between photoreceptors in the intact adult retina. Conversely, while photoreceptors are capable of releasing EVs, at least in culture, these are taken up by glia and not by retinal neurons. Our findings provide the first evidence of functional NT-like processes forming between sensory neurons in culture and in vivo.
Subject(s)
Extracellular Vesicles , Nanotubes , Animals , Cell Communication , Mammals , Neurons , RetinaABSTRACT
In development, almost all stratified neurons must migrate from their birthplace to the appropriate neural layer. Photoreceptors reside in the most apical layer of the retina, near their place of birth. Whether photoreceptors require migratory events for fine-positioning and/or retention within this layer is not well understood. Here, we show that photoreceptor nuclei of the developing mouse retina cyclically exhibit rapid, dynein-1-dependent translocation toward the apical surface, before moving more slowly in the basal direction, likely due to passive displacement by neighboring retinal nuclei. Attenuating dynein 1 function in rod photoreceptors results in their ectopic basal displacement into the outer plexiform layer and inner nuclear layer. Synapse formation is also compromised in these displaced cells. We propose that repeated, apically directed nuclear translocation events are necessary to ensure retention of post-mitotic photoreceptors within the emerging outer nuclear layer during retinogenesis, which is critical for correct neuronal lamination.
Subject(s)
Cell Nucleus/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Retina/metabolism , Actomyosin/metabolism , Animals , Dyneins/metabolism , Kinetics , Mice, Transgenic , Microtubules/metabolism , Myosin Type II/metabolism , Neurogenesis , Polymerization , Protein Transport , Retinal Cone Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Synapses/metabolismABSTRACT
Gliosis is a complex process comprising upregulation of intermediate filament (IF) proteins, particularly glial fibrillary acidic protein (GFAP) and vimentin, changes in glial cell morphology (hypertrophy) and increased deposition of inhibitory extracellular matrix molecules. Gliosis is common to numerous pathologies and can have deleterious effects on tissue function and regeneration. The role of IFs in gliosis is controversial, but a key hypothesized function is the stabilization of glial cell hypertrophy. Here, we developed RNAi approaches to examine the role of GFAP and vimentin in vivo in a murine model of inherited retinal degeneration, the Rhodopsin knockout (Rho-/- ) mouse. Specifically, we sought to examine the role of these IFs in the establishment of Müller glial hypertrophy during progressive degeneration, as opposed to (more commonly assessed) acute injury. Prevention of Gfap upregulation had a significant effect on the morphology of reactive Müller glia cells in vivo and, more strikingly, the reduction of Vimentin expression almost completely prevented these cells from undergoing degeneration-associated hypertrophy. Moreover, and in contrast to studies in knockout mice, simultaneous suppression of both GFAP and vimentin expression led to severe changes in the cytoarchitecture of the retina, in both diseased and wild-type eyes. These data demonstrate a crucial role for Vimentin, as well as GFAP, in the establishment of glial hypertrophy and support the further exploration of RNAi-mediated knockdown of vimentin as a potential therapeutic approach for modulating scar formation in the degenerating retina.
Subject(s)
Ependymoglial Cells , Glial Fibrillary Acidic Protein , Retinal Degeneration , Vimentin , Animals , Ependymoglial Cells/metabolism , Glial Fibrillary Acidic Protein/metabolism , Hypertrophy/metabolism , Hypertrophy/pathology , Intermediate Filaments/metabolism , Mice , Neuroglia/metabolism , RNA Interference , Retina/metabolism , Retinal Degeneration/pathology , Vimentin/metabolismABSTRACT
Age-related macular degeneration and other macular diseases result in the loss of light-sensing cone photoreceptors, causing irreversible sight impairment. Photoreceptor replacement may restore vision by transplanting healthy cells, which must form new synaptic connections with the recipient retina. Despite recent advances, convincing evidence of functional connectivity arising from transplanted human cone photoreceptors in advanced retinal degeneration is lacking. Here, we show restoration of visual function after transplantation of purified human pluripotent stem cell-derived cones into a mouse model of advanced degeneration. Transplanted human cones elaborate nascent outer segments and make putative synapses with recipient murine bipolar cells (BCs), which themselves undergo significant remodeling. Electrophysiological and behavioral assessments demonstrate restoration of surprisingly complex light-evoked retinal ganglion cell responses and improved light-evoked behaviors in treated animals. Stringent controls exclude alternative explanations, including material transfer and neuroprotection. These data provide crucial validation for photoreceptor replacement therapy and for the potential to rescue cone-mediated vision.
Subject(s)
Induced Pluripotent Stem Cells/metabolism , Macular Degeneration/therapy , Organoids/transplantation , Recovery of Function/physiology , Retinal Cone Photoreceptor Cells/metabolism , Animals , Biomarkers/metabolism , Cell Differentiation , Dependovirus/genetics , Dependovirus/metabolism , Disease Models, Animal , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Macular Degeneration/genetics , Macular Degeneration/metabolism , Macular Degeneration/pathology , Male , Mice , Mice, Transgenic , Mycotoxins/genetics , Mycotoxins/metabolism , Organoids/cytology , Organoids/metabolism , Peripherins/genetics , Peripherins/metabolism , Photic Stimulation , Primary Cell Culture , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Receptors, Glutamate/genetics , Receptors, Glutamate/metabolism , Retinal Bipolar Cells/cytology , Retinal Bipolar Cells/metabolism , Retinal Cone Photoreceptor Cells/cytology , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/metabolism , Synapses/metabolism , Transplantation, Heterologous , Vision, Ocular/physiologyABSTRACT
WHAT IS KNOWN AND OBJECTIVE: Pharmacogenomic biomarkers are now used in many clinical care settings and represent one of the successes of precision medicine. Genetic variants are associated with pharmacokinetic and pharmacodynamic changes leading to medication adverse effects and changes in clinical response. Actionable pharmacogenomic variants are common in transplant recipients and have implications for medications used in transplant, but yet are not broadly incorporated into practice. METHODS: From the Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacogenetics Working Group guidelines, and PharmGKB databases, 12 pharmacogenomic genes with 30 variants were selected and used to create diplotypes and actionable pharmacogenomic phenotypes. A total of 853 kidney allograft recipients who had genomic information available from a genome-wide association study were included. RESULTS: Each recipient had at least one actionable pharmacogenomic diplotype/phenotype, whereas the majority (58%) had three or four actionable diplotypes/phenotypes and 17.4% had five or more among the 12 genes. The participants carried actionable diplotypes/phenotypes for multiple medications, including tacrolimus, azathioprine, clopidogrel, warfarin, simvastatin, voriconazole, antidepressants and proton-pump inhibitors. WHAT IS NEW AND CONCLUSION: Pharmacogenomic variants are common in transplant recipients, and transplant recipients receive medications that have actionable variants. CLINICAL TRIAL: Genomics of Transplantation, clinicaltrials.gov (NCT01714440).
Subject(s)
Kidney Transplantation/methods , Pharmacogenetics/methods , Pharmacogenomic Variants , Adult , Female , Genome-Wide Association Study , Humans , Male , Middle Aged , Phenotype , Prospective StudiesABSTRACT
Retinal degeneration is a leading cause of untreatable blindness in the industrialised world. It is typically irreversible and there are few curative treatments available. The use of stem cells to generate new retinal neurons for transplantation purposes has received significant interest in recent years and is beginning to move towards clinical trials. However, such approaches are likely to be most effective for relatively focal areas of repair. An intriguing complementary approach is endogenous self-repair. Retinal cells from the ciliary marginal zone (CMZ), retinal pigment epithelium (RPE) and Müller glial cells (MG) have all been shown to play a role in retinal repair, typically in lower vertebrates. Among them, MG have received renewed interest, due to their distribution throughout (centre to periphery) the neural retina and their potential to re-acquire a progenitor-like state following retinal injury with the ability to proliferate and generate new neurons. Triggering these innate self-repair mechanisms represents an exciting therapeutic option in treating retinal degeneration. However, these cells behave differently in mammalian and non-mammalian species, with a considerably restricted potential in mammals. In this short review, we look at some of the recent progress made in our understanding of the signalling pathways that underlie MG-mediated regeneration in lower vertebrates, and some of the challenges that have been revealed in our attempts to reactivate this process in the mammalian retina.
Subject(s)
Ependymoglial Cells/physiology , Neuroglia/physiology , Regeneration/physiology , Retinal Degeneration/physiopathology , Retinal Neurons/physiology , Animals , Humans , Retinal Degeneration/pathologyABSTRACT
Irreversible photoreceptor cell death is a major cause of blindness in many retinal dystrophies. A better understanding of the molecular mechanisms underlying the progressive loss of photoreceptor cells remains therefore crucial. Abnormal expression of microRNAs (miRNAs) has been linked with the aetiology of a number of retinal dystrophies. However, their role during the degenerative process remains poorly understood. Loss of cone photoreceptors in the human macula has the greatest impact on sight as these cells provide high acuity vision. Using a Chrnb4-cre; Dicerflox/flox conditional knockout mouse (Dicer CKO) to delete Dicer1 from cone cells, we show that cone photoreceptor cells degenerate and die in the Dicer-deleted retina. Embryonic eye morphogenesis appeared normal in Dicer CKO mice. Cone photoreceptor abnormalities were apparent by 3 weeks of age, displaying either very short or absent outer segments. By 4 months 50% of cones were lost and cone function was impaired as assessed by electroretinography (ERG). RNAseq analysis of the Dicer CKO retina revealed altered expression of genes involved in the visual perception pathway. These data show that loss of Dicer1 leads to early-onset cone cell degeneration and suggest that Dicer1 is essential for cone photoreceptor survival and homeostasis.
Subject(s)
Cell Death/physiology , Color Vision/physiology , DEAD-box RNA Helicases/metabolism , Integrases/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Nicotinic/metabolism , Retinal Cone Photoreceptor Cells/cytology , Retinal Cone Photoreceptor Cells/metabolism , Ribonuclease III/metabolism , Visual Acuity/physiology , Animals , Cell Death/genetics , Color Vision/genetics , DEAD-box RNA Helicases/genetics , Electroretinography , Female , Integrases/genetics , Male , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Receptors, Nicotinic/genetics , Ribonuclease III/genetics , Visual Acuity/geneticsABSTRACT
The eye often leads the way in the development of novel strategies for treating neurodegenerative disorders. Here, we highlight an exciting new study examining the potential for reactivating endogenous repair mechanisms and enabling the mammalian retina to repair itself.
Subject(s)
Photoreceptor Cells/physiology , Regeneration/physiology , Retina/physiology , Retinal Degeneration/physiopathology , Visual Perception/physiology , Animals , Cell Proliferation/genetics , Ependymoglial Cells/physiology , Humans , Regeneration/genetics , Retina/physiopathology , Retinal Degeneration/genetics , Retinal Degeneration/therapyABSTRACT
Within the mammalian retina, both Müller glia and astrocytes display reactivity in response to many forms of retinal injury and disease in a process termed gliosis. Reactive gliosis is a complex process that is considered to represent a cellular response to protect the retina from further damage and to promote its repair following pathological insult. It includes morphological, biochemical and physiological changes, which may vary depending on the type and degree of the initial injury. Not only does gliosis have numerous triggers, but also there is a great degree of heterogeneity in the glial response, creating multiple levels of complexity. For these reasons, understanding the process of glial scar formation and how this process differs in different pathological conditions and finding strategies to circumvent these barriers represent major challenges to the advancement of many ocular therapies.
Subject(s)
Ependymoglial Cells/physiology , Gliosis/pathology , Retinal Diseases/pathology , Animals , Astrocytes/physiology , Cicatrix/pathology , Cytokines/metabolism , Gliosis/complications , Gliosis/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Intermediate Filament Proteins/metabolism , Photoreceptor Cells, Vertebrate/transplantation , Retina/injuries , Retina/metabolism , Retina/pathology , Retinal Detachment/etiology , Retinal Detachment/prevention & control , Retinal Diseases/metabolism , Species Specificity , Vertebrates/physiologyABSTRACT
The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage disorders characterized by general neurodegeneration and premature death. Sight loss is also a major symptom in NCLs, severely affecting the quality of life of patients, but it is not targeted effectively by brain-directed therapies. Here we set out to explore the therapeutic potential of an ocular gene therapy to treat sight loss in NCL due to a deficiency in the transmembrane protein CLN6. We found that, although Cln6nclf mice presented mainly with photoreceptor degeneration, supplementation of CLN6 in photoreceptors was not beneficial. Because the level of CLN6 is low in photoreceptors but high in bipolar cells (retinal interneurons that are only lost in Cln6-deficient mice at late disease stages), we explored the therapeutic effects of delivering CLN6 to bipolar cells using adeno-associated virus (AAV) serotype 7m8. Bipolar cell-specific expression of CLN6 slowed significantly the loss of photoreceptor function and photoreceptor cells. This study shows that the deficiency of a gene normally expressed in bipolar cells can cause the loss of photoreceptors and that this can be prevented by bipolar cell-directed treatment.
Subject(s)
Membrane Proteins/genetics , Neuronal Ceroid-Lipofuscinoses/genetics , Photoreceptor Cells/metabolism , Retinal Bipolar Cells/metabolism , Animals , Dependovirus/genetics , Disease Models, Animal , Gene Expression , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors/genetics , Humans , Immunohistochemistry , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Neuronal Ceroid-Lipofuscinoses/metabolism , Neuronal Ceroid-Lipofuscinoses/pathology , Neuronal Ceroid-Lipofuscinoses/therapy , Photoreceptor Cells/pathologyABSTRACT
Adeno-associated viral vectors are showing great promise as gene therapy vectors for a wide range of retinal disorders. To date, evaluation of therapeutic approaches has depended almost exclusively on the use of animal models. With recent advances in human stem cell technology, stem cell-derived retina now offers the possibility to assess efficacy in human organoids in vitro. Here we test six adeno-associated virus (AAV) serotypes [AAV2/2, AAV2/9, AAV2/8, AAV2/8T(Y733F), AAV2/5, and ShH10] to determine their efficiency in transducing mouse and human pluripotent stem cell-derived retinal pigment epithelium (RPE) and photoreceptor cells in vitro. All the serotypes tested were capable of transducing RPE and photoreceptor cells in vitro. AAV ShH10 and AAV2/5 are the most efficient vectors at transducing both mouse and human RPE, while AAV2/8 and ShH10 achieved similarly robust transduction of human embryonic stem cell-derived cone photoreceptors. Furthermore, we show that human embryonic stem cell-derived photoreceptors can be used to establish promoter specificity in human cells in vitro. The results of this study will aid capsid selection and vector design for preclinical evaluation of gene therapy approaches, such as gene editing, that require the use of human cells and tissues.
Subject(s)
Dependovirus/physiology , Genetic Vectors/genetics , Photoreceptor Cells/cytology , Photoreceptor Cells/metabolism , Pluripotent Stem Cells/cytology , Retinal Pigment Epithelium/cytology , Viral Tropism , Animals , Cell Differentiation , Cells, Cultured , Dependovirus/classification , Fluorescent Antibody Technique , Gene Expression , Gene Transfer Techniques , Genes, Reporter , Humans , Mice , Organ Specificity/genetics , Promoter Regions, Genetic , Transduction, Genetic , TransgenesABSTRACT
Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl-/- and Prph2rd2/rd2, but not Nrl-/-;RPE65R91W/R91W, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome.
Subject(s)
Blindness/therapy , Retinal Cone Photoreceptor Cells/transplantation , Retinal Degeneration/therapy , Stem Cell Transplantation , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Blindness/genetics , Blindness/pathology , Cell Differentiation/genetics , Disease Models, Animal , Eye Proteins/genetics , Humans , Mice , Peripherins/genetics , Retina/pathology , Retina/transplantation , Retinal Cone Photoreceptor Cells/cytology , Retinal Degeneration/pathology , Stem Cells/cytology , cis-trans-Isomerases/geneticsABSTRACT
Transplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.
Subject(s)
Pluripotent Stem Cells/cytology , Retinal Cone Photoreceptor Cells/cytology , Retinal Cone Photoreceptor Cells/transplantation , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Differentiation , Cells, Cultured , Disease Models, Animal , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/ultrastructure , Humans , Pluripotent Stem Cells/metabolism , Retinal Degeneration/pathology , Retinal Degeneration/therapy , Time FactorsABSTRACT
PURPOSE OF REVIEW: A major cause of visual disorders is dysfunction and/or loss of the light-sensitive cells of the retina, the photoreceptors. To develop better treatments for patients, we need to understand how inherited retinal disease mutations result in the dysfunction of photoreceptors. New advances in the field of stem cell and gene editing research offer novel ways to model retinal dystrophies in vitro and present opportunities to translate basic biological insights into therapies. This brief review will discuss some of the issues that should be taken into account when carrying out disease modelling and gene editing of retinal cells. We will discuss (i) the use of human induced pluripotent stem cells (iPSCs) for disease modelling and cell therapy; (ii) the importance of using isogenic iPSC lines as controls; (iii) CRISPR/Cas9 gene editing of iPSCs; and (iv) in vivo gene editing using AAV vectors. RECENT FINDINGS: Ground-breaking advances in differentiation of iPSCs into retinal organoids and methods to derive mature light sensitive photoreceptors from iPSCs. Furthermore, single AAV systems for in vivo gene editing have been developed which makes retinal in vivo gene editing therapy a real prospect. SUMMARY: Genome editing is becoming a valuable tool for disease modelling and in vivo gene editing in the retina.
ABSTRACT
Age-related macular degeneration and inherited retinal degenerations represent the leading causes of blindness in industrialized countries. Despite different initiating causes, they share a common final pathophysiology, the loss of the light sensitive photoreceptors. Replacement by transplantation may offer a potential treatment strategy for both patient populations. The last decade has seen remarkable progress in our ability to generate retinal cell types, including photoreceptors, from a variety of murine and human pluripotent stem cell sources. Driven in large part by the requirement for renewable cell sources, stem cells have emerged not only as a promising source of replacement photoreceptors but also to provide in vitro systems with which to study retinal development and disease processes and to test therapeutic agents.
Subject(s)
Pluripotent Stem Cells/cytology , Retinal Degeneration/therapy , Stem Cell Transplantation , Animals , Humans , Mice , Retina/cytology , Retina/physiopathologyABSTRACT
The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor ß2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1-/- mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.