RESUMO
Multipotent retinal progenitor cells (RPCs) generate various cell types in a precise chronological order, but how exactly cone photoreceptor production is restricted to early stages remains unclear. Here, we show that the POU-homeodomain factors Pou2f1/Pou2f2, the homologs of Drosophila temporal identity factors nub/pdm2, regulate the timely production of cones in mice. Forcing sustained expression of Pou2f1 or Pou2f2 in RPCs expands the period of cone production, whereas misexpression in late-stage RPCs triggers ectopic cone production at the expense of late-born fates. Mechanistically, we report that Pou2f1 induces Pou2f2 expression, which binds to a POU motif in the promoter of the rod-inducing factor Nrl to repress its expression. Conversely, conditional inactivation of Pou2f2 in RPCs increases Nrl expression and reduces cone production. Finally, we provide evidence that Pou2f1 is part of a cross-regulatory cascade with the other temporal identity factors Ikzf1 and Casz1. These results uncover Pou2f1/2 as regulators of the temporal window for cone genesis and, given their widespread expression in the nervous system, raise the possibility of a general role in temporal patterning.This article has an associated 'The people behind the papers' interview.
Assuntos
Proteínas do Olho/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Fator 2 de Transcrição de Octâmero/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Drosophila/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células-Tronco/metabolismoRESUMO
Retinal diseases constitute a genetically and phenotypically diverse group of clinical conditions leading to vision impairment or blindness with limited treatment options. Advances in reprogramming of somatic cells to induced pluripotent stem cells and generation of three-dimensional organoids resembling the native retina offer promising tools to interrogate disease mechanisms and evaluate potential therapies for currently incurable retinal neurodegeneration. Next-generation sequencing, single-cell analysis, advanced electrophysiology, and high-throughput screening approaches are expected to greatly expand the utility of stem cell-derived retinal cells and organoids for developing personalized treatments. In this review, we discuss the current status and future potential of combining retinal organoids as human models with recent technologies to advance the development of gene, cell, and drug therapies for retinopathies.
Assuntos
Modelos Biológicos , Organoides/patologia , Células-Tronco Pluripotentes/citologia , Retina/patologia , Animais , Diferenciação Celular , Terapia Genética , HumanosRESUMO
Doublecortin (DCX) is a protein needed for cortical development, and DCX mutations cause cortical malformations in humans. The microtubule-binding activity of DCX is well-described and is important for its function, such as supporting neuronal migration and dendrite growth during development. Previous work showed that microtubule binding is not sufficient for DCX-mediated promotion of dendrite growth and that domains in DCX's C terminus are also required. The more C-terminal regions of DCX bind several other proteins, including the adhesion receptor neurofascin and clathrin adaptors. We recently identified a role for DCX in endocytosis of neurofascin. The disease-associated DCX-G253D mutant protein is known to be deficient in binding neurofascin, and we now asked if disruption of neurofascin endocytosis underlies the DCX-G253D-associated pathology. We first demonstrated that DCX functions in endocytosis as a complex with both the clathrin adaptor AP-2 and neurofascin: disrupting either clathrin adaptor binding (DCX-ALPA) or neurofascin binding (DCX-G253D) decreased neurofascin endocytosis in primary neurons. We then investigated a known function for DCX, namely, increasing dendrite growth in cultured neurons. Surprisingly, we found that the DCX-ALPA and DCX-G253D mutants yield distinct dendrite phenotypes. Unlike DCX-ALPA, DCX-G253D caused a dominant-negative dendrite growth phenotype. The endocytosis defect of DCX-G253D thus was separable from its detrimental effects on dendrite growth. We recently identified Dcx-R59H as a dominant allele and can now classify Dcx-G253D as a second Dcx allele that acts dominantly to cause pathology, but does so via a different mechanism.
Assuntos
Dendritos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Neurônios/citologia , Neuropeptídeos/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Animais , Sítios de Ligação , Células COS , Moléculas de Adesão Celular/metabolismo , Chlorocebus aethiops , Dendritos/genética , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Endocitose/genética , Células HEK293 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , RatosRESUMO
PURPOSE: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.
Assuntos
Diferenciação Celular , Diterpenos/farmacologia , Células-Tronco Embrionárias Humanas/citologia , Organoides/citologia , Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Retinaldeído/farmacologia , Transcriptoma/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Transcriptoma/efeitos dos fármacosRESUMO
Doublecortin on the X-chromosome (DCX) is a neuronal microtubule-binding protein with a multitude of roles in neurodevelopment. In humans, DCX is a major genetic locus for X-linked lissencephaly. The best studied defects are in neuronal migration during corticogenesis and in the hippocampus, as well as axon and dendrite growth defects. Much effort has been directed at understanding the molecular and cellular bases of DCX-linked lissencephaly. The focus has been in particular on defects in microtubule assembly and bundling, using knock-out mice and expression of WT and mutant Dcx in non-neuronal cells. Dcx also binds other proteins besides microtubules, such as spinophilin (abbreviated spn; gene name Ppp1r9b protein phosphatase 1 regulatory subunit 9b) and the clathrin adaptors AP-1 and AP-2. Even though many non-sense and missense mutations of Dcx are known, their molecular and cellular defects are still only incompletely understood. It is also largely unknown how neurons are affected by expression of DCX patient alleles. We have now characterized several patient DCX alleles (DCX-R89G, DCX-R59H, DCX-246X, DCX-272X, and DCX-303X) using a gain-of-function dendrite growth assay in cultured rat neurons in combination with the determination of molecular binding activities and subcellular localization in non-neuronal and neuronal cells. First, we find that several mutants (Dcx-R89G and Dcx-272X) were loss-of-function alleles (as had been postulated) but surprisingly acted via different cellular mechanisms. Second, one allele (Dcx-R59H) formed cytoplasmic aggregates, which contained Hspa1B (heat shock protein 1B hsp70) and ubiquitinated proteins, trapped other cytoskeletal proteins, including spinophilin, and led to increased autophagy. This allele could thus be categorized as "off-pathway"/possibly neomorph. Our findings thus suggested that distinct DCX alleles caused dysfunction by different mechanisms.
Assuntos
Hipocampo/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mutação/genética , Neurônios/patologia , Neuropeptídeos/metabolismo , Alelos , Animais , Movimento Celular , Células Cultivadas , Dendritos/metabolismo , Dendritos/patologia , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Hipocampo/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Mutagênese Sítio-Dirigida , Neurogênese , Neurônios/metabolismo , Neuropeptídeos/genética , Fenótipo , RatosRESUMO
Doublecortin on X chromosome (DCX) is one of two major genetic loci underlying human lissencephaly, a neurodevelopmental disorder with defects in neuronal migration and axon outgrowth. DCX is a microtubule-binding protein, and much work has focused on its microtubule-associated functions. DCX has other reported binding partners, including the cell adhesion molecule neurofascin, but the functional significance of the DCX-neurofascin interaction is not understood. Neurofascin localizes strongly to the axon initial segment in mature neurons, where it plays a role in assembling and maintaining other axon initial segment components. During development, neurofascin likely plays additional roles in axon guidance and in GABAergic synaptogenesis. We show here that DCX can modulate the surface distribution of neurofascin in developing cultured rat neurons and thereby the relative extent of accumulation between the axon initial segment and soma and dendrites. Mechanistically, DCX acts via increasing endocytosis of neurofascin from soma and dendrites. Surprisingly, DCX increases neurofascin endocytosis apparently independently of its microtubule-binding activity. We additionally show that the patient allele DCXG253D still binds microtubules but is deficient in promoting neurofascin endocytosis. We propose that DCX acts as an endocytic adaptor for neurofascin to fine-tune its surface distribution during neuronal development.
Assuntos
Moléculas de Adesão Celular/metabolismo , Endocitose/fisiologia , Proteínas Associadas aos Microtúbulos/farmacologia , Microtúbulos/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/fisiologia , Neuropeptídeos/farmacologia , Animais , Anquirinas/metabolismo , Moléculas de Adesão Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Polaridade Celular/genética , Células Cultivadas , Chlorocebus aethiops , Dendritos/metabolismo , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Imunoprecipitação , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/genética , Fatores de Crescimento Neural/genética , Neurônios/citologia , Neuropeptídeos/genética , Mutação Puntual/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Interferente Pequeno/metabolismo , Ratos , Canais de Sódio/metabolismo , Estatísticas não Paramétricas , Fatores de Tempo , TransfecçãoRESUMO
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.
Assuntos
Ciliopatias , Retina , Humanos , Retina/metabolismo , Ciliopatias/genética , Ciliopatias/terapia , Ciliopatias/metabolismo , Mutação , Cílios/metabolismo , Células-Tronco/metabolismoRESUMO
Mutations in the IQ calmodulin-binding motif containing B1 (IQCB1)/NPHP5 gene encoding the ciliary protein nephrocystin 5 cause early-onset blinding disease Leber congenital amaurosis (LCA), together with kidney dysfunction in Senior-Løken syndrome. For in vitro disease modeling, we obtained dermal fibroblasts from patients with NPHP5-LCA that were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into retinal pigment epithelium (RPE) and retinal organoids. Patient fibroblasts and RPE demonstrated aberrantly elongated ciliary axonemes. Organoids revealed impaired development of outer segment structures, which are modified primary cilia, and mislocalization of visual pigments to photoreceptor cell soma. All patient-derived cells showed reduced levels of CEP290 protein, a critical cilia transition zone component interacting with NPHP5, providing a plausible mechanism for aberrant ciliary gating and cargo transport. Disease phenotype in NPHP5-LCA retinal organoids could be rescued by adeno-associated virus (AAV)-mediated IQCB1/NPHP5 gene augmentation therapy. Our studies thus establish a human disease model and a path for treatment of NPHP5-LCA.
Assuntos
Calmodulina , Ciliopatias , Antígenos de Neoplasias/genética , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciliopatias/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Mutação , Pigmentos da Retina/metabolismoRESUMO
Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.
Assuntos
Proteínas de Homeodomínio/genética , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Organoides/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Transativadores/genética , Adulto , Diferenciação Celular , Criança , Pré-Escolar , Dependovirus , Feminino , Terapia Genética/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Amaurose Congênita de Leber/patologia , Modelos Biológicos , Mutação , Opsinas/metabolismo , Organoides/citologia , Fenótipo , Retina/citologia , Análise de Sequência de RNA , Análise de Célula Única , TranscriptomaRESUMO
Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Nanofibras/química , Epitélio Pigmentado da Retina/citologia , Proteínas de Soja/química , Alicerces Teciduais/química , Linhagem Celular , Módulo de Elasticidade , Perfilação da Expressão Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nanofibras/ultraestrutura , Poliésteres/química , Epitélio Pigmentado da Retina/ultraestrutura , Proteínas de Soja/ultraestruturaRESUMO
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.
Assuntos
Dependovirus/fisiologia , Vetores Genéticos/genética , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Células-Tronco Pluripotentes/citologia , Epitélio Pigmentado da Retina/citologia , Tropismo Viral , Animais , Diferenciação Celular , Células Cultivadas , Dependovirus/classificação , Imunofluorescência , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Camundongos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Transdução Genética , TransgenesRESUMO
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.
Assuntos
Cegueira/terapia , Células Fotorreceptoras Retinianas Cones/transplante , Degeneração Retiniana/terapia , Transplante de Células-Tronco , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Cegueira/genética , Cegueira/patologia , Diferenciação Celular/genética , Modelos Animais de Doenças , Proteínas do Olho/genética , Humanos , Camundongos , Periferinas/genética , Retina/patologia , Retina/transplante , Células Fotorreceptoras Retinianas Cones/citologia , Degeneração Retiniana/patologia , Células-Tronco/citologia , cis-trans-Isomerases/genéticaRESUMO
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.
Assuntos
Células-Tronco Pluripotentes/citologia , Degeneração Retiniana/terapia , Transplante de Células-Tronco , Animais , Humanos , Camundongos , Retina/citologia , Retina/fisiopatologiaRESUMO
Loss of cone photoreceptors, crucial for daylight vision, has the greatest impact on sight in retinal degeneration. Transplantation of stem cell-derived L/M-opsin cones, which form 90% of the human cone population, could provide a feasible therapy to restore vision. However, transcriptomic similarities between fetal and stem cell-derived cones remain to be defined, in addition to development of cone cell purification strategies. Here, we report an analysis of the human L/M-opsin cone photoreceptor transcriptome using an AAV2/9.pR2.1:GFP reporter. This led to the identification of a cone-enriched gene signature, which we used to demonstrate similar gene expression between fetal and stem cell-derived cones. We then defined a cluster of differentiation marker combination that, when used for cell sorting, significantly enriches for cone photoreceptors from the fetal retina and stem cell-derived retinal organoids, respectively. These data may facilitate more efficient isolation of human stem cell-derived cones for use in clinical transplantation studies.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Opsinas de Bastonetes/genética , Transcriptoma/genética , Diferenciação Celular/genética , Feto/citologia , Feto/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Retina/crescimento & desenvolvimento , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/transplante , Degeneração Retiniana/patologiaRESUMO
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.
Assuntos
Células-Tronco Embrionárias Murinas/transplante , Células Fotorreceptoras Retinianas Cones/citologia , Degeneração Retiniana/terapia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Fator 6 Nuclear de Hepatócito/metabolismo , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Opsinas/metabolismo , Receptores Nucleares Órfãos/antagonistas & inibidores , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Fatores de Transcrição Otx/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/patologia , Transdução de Sinais , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
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.