RESUMO
PURPOSE: The generation of three-dimensional (3D) organoids with optic cup-like structures from pluripotent stem cells has created opportunities for investigating mammalian retinal development in vitro. However, retinal organoids in culture do not completely reflect the developmental state and in vivo architecture of the rod-dominant mouse retina. The goals of this study were to develop an efficient protocol for generating retinal organoids from stem cells and examine the morphogenesis of rods in vitro. METHODS: To assess rod photoreceptor differentiation in retinal organoids, we took advantage of Nrl-green fluorescent protein (GFP) mice that show rod-specific expression of GFP directed by the promoter of leucine zipper transcription factor NRL. Using embryonic and induced pluripotent stem cells (ESCs and iPSCs, respectively) derived from the Nrl-GFP mouse, we were successful in establishing long-term retinal organoid cultures using modified culture conditions (called High Efficiency Hypoxia Induced Generation of Photoreceptors in Retinal Organoids, or HIPRO). RESULTS: We demonstrated efficient differentiation of pluripotent stem cells to retinal structures. More than 70% of embryoid bodies formed optic vesicles at day (D) 7, >50% produced optic cups by D10, and most of them survived until at least D35. The HIPRO organoids included distinct inner retina neurons in a somewhat stratified architecture and mature Müller glia spanning the entire retina. Almost 70% of the cells in the retinal organoids were rod photoreceptors that exhibited elongated cilia. Transcriptome profiles of GFP+ rod photoreceptors, purified from organoids at D25-35, demonstrated a high correlation with the gene profiles of purified rods from the mouse retina at P2 to P6, indicating their early state of differentiation. CONCLUSIONS: The 3D retinal organoids, generated by HIPRO method, closely mimic in vivo retinogenesis and provide an efficient in vitro model to investigate photoreceptor development and modeling disease pathology.
RESUMO
Ciliopathies display extensive genetic and clinical heterogeneity, varying in severity, age of onset, disease progression and organ systems affected. Retinal involvement, as demonstrated by photoreceptor dysfunction or death, is a highly penetrant phenotype among a vast majority of ciliopathies. Photoreceptor cells possess a specialized and modified sensory cilium with membrane discs where efficient photon capture and ensuing signaling cascade initiate the visual process. Disruptions of cilia biogenesis and protein transport lead to impairment of photoreceptor function and eventually degeneration. Despite advances in elucidation of ciliogenesis and photoreceptor cilia defects, we have limited understanding of pathogenic mechanisms underlying retinal phenotype(s) observed in human ciliopathies. Patient-derived induced pluripotent stem cell (iPSC)-based approaches offer a unique opportunity to complement studies with model organisms and examine cilia disease relevant to humans. Three-dimensional retinal organoids from iPSC lines feature laminated cytoarchitecture, apical-basal polarity and emergence of a ciliary structure, thereby permitting pathogenic modeling of human photoreceptors in vitro. Here, we review the biology of photoreceptor cilia and associated defects and discuss recent progress in evolving treatment modalities, especially using patient-derived iPSCs, for retinal ciliopathies.
RESUMO
Numerous degenerative diseases affecting visual function, including glaucoma and retinitis pigmentosa, are produced by the loss of different types of retinal cells. Cell replacement therapy has emerged as a promising strategy for treating these and other retinal diseases. The retinal margin or ciliary body (CB) of mammals has been proposed as a potential source of cells to be used in degenerative conditions affecting the retina because it has been reported it might hold neurogenic potential beyond embryonic development. However, many aspects of the origin and biology of the CB are unknown and more recent experiments have challenged the capacity of CB cells to generate different types of retinal neurons. Here we review the most recent findings about the development of the marginal zone of the retina in different vertebrates and some of the mechanisms underlying the proliferative and neurogenic capacity of this fascinating region of the vertebrates eye. In addition, we performed experiments to isolate CB cells from the mouse retina, generated neurospheres and observed that they can be expanded with a proliferative ratio similar to neural stem cells. When induced to differentiate, cells derived from the CB neurospheres start to express early neural markers but, unlike embryonic stem cells, they are not able to fully differentiate in vitro or generate retinal organoids. Together with previous reports on the neurogenic capacity of CB cells, also reviewed here, our results contribute to the current knowledge about the potentiality of this peripheral region of the eye as a therapeutic source of functional retinal neurons in degenerative diseases.
Assuntos
Técnicas de Cultura de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Corpo Ciliar/citologia , Neurogênese/fisiologia , Degeneração Retiniana/terapia , Animais , Diferenciação Celular , Células Cultivadas , Corpo Ciliar/fisiologia , Humanos , CamundongosRESUMO
Mutations in CEP290 cause ciliogenesis defects, leading to diverse clinical phenotypes, including Leber congenital amaurosis (LCA). Gene therapy for CEP290-associated diseases is hindered by the 7.4 kb CEP290 coding sequence, which is difficult to deliver in vivo. The multi-domain structure of the CEP290 protein suggests that a specific CEP290 domain may complement disease phenotypes. Thus, we constructed AAV vectors with overlapping CEP290 regions and evaluated their impact on photoreceptor degeneration in Cep290rd16/rd16 and Cep290rd16/rd16;Nrl-/- mice, two models of CEP290-LCA. One CEP290 fragment (the C-terminal 989 residues, including the domain deleted in mutant mice) reconstituted CEP290 function and resulted in cone preservation and delayed rod death. The CEP290 C-terminal domain also improved cilia phenotypes in mouse embryonic fibroblasts and iPSC-derived retinal organoids carrying the Cep290rd16 mutation. Our study strongly argues for in trans complementation of CEP290 mutations by a cognate fragment and suggests therapeutic avenues.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Terapia Genética , Amaurose Congênita de Leber/terapia , Mutação , Retina/citologia , Degeneração Retiniana/terapia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Cílios/metabolismo , Cílios/patologia , Dependovirus/genética , Modelos Animais de Doenças , Proteínas do Olho/fisiologia , Feminino , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/patologia , Masculino , Camundongos , Camundongos Knockout , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones , Degeneração Retiniana/genética , Degeneração Retiniana/patologiaRESUMO
Pluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV) bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25) reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.