RESUMEN
Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Müller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in Crb1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Müller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Ependimogliales/metabolismo , Proteínas del Ojo/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Organoides/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Adulto , Proteínas Portadoras/genética , Células Cultivadas , Dependovirus/genética , Células Ependimogliales/citología , Células Ependimogliales/ultraestructura , Proteínas del Ojo/genética , Femenino , Feto , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Proteínas de la Membrana/genética , Microscopía Inmunoelectrónica , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/genética , Organoides/citología , Células Fotorreceptoras de Vertebrados/ultraestructura , Embarazo , Retina/citología , Retina/embriologíaRESUMEN
In vitro retinal organoid modeling from human pluripotent stem cells is becoming more common place in many ophthalmic laboratories worldwide. These organoids mimic human retinogenesis through formation of organized layered retinal structures that display markers for typical retinal cell types. Pivotally these humanized retinal models provide a stepping stone to the clinic as therapeutic tools and are expected to provide a promising alternative to current animal models. Thus pluripotent stem cell based healthy as well as diseased human retinal organoids are attractive for use in drug potency assays and gene augmentation therapeutics. Here we outline an established protocol for generation of these retinal organoids and how they can be used in conjunction with adeno-associated virus vectors for transgene expression assays.