RESUMEN
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.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Macular/terapia , Organoides/trasplante , Recuperación de la Función/fisiología , Células Fotorreceptoras Retinianas Conos/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Masculino , Ratones , Ratones Transgénicos , Micotoxinas/genética , Micotoxinas/metabolismo , Organoides/citología , Organoides/metabolismo , Periferinas/genética , Periferinas/metabolismo , Estimulación Luminosa , Cultivo Primario de Células , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Células Bipolares de la Retina/citología , Células Bipolares de la Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/citología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Sinapsis/metabolismo , Trasplante Heterólogo , Visión Ocular/fisiologíaRESUMEN
Extracellular ATP-induced Ca2+ signalling is critical in regulating diverse physiological and disease processes. Emerging evidence suggests high concentrations of extracellular ATP in tumour tissues. In this study, we examined the P2 receptor for ATP-induced Ca2+ signalling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) showed that extracellular ATP induced an increase in the [Ca2+]i in human HCC Huh-7 and HepG2 cells. NF546, a P2Y11 receptor agonist was equally effective in inducing an increase in the [Ca2+]i. In contrast, agonists for the P2X receptors (αßmeATP and BzATP), P2Y1 receptor (MRS2365) or P2Y2 receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increases in the [Ca2+]i were strongly inhibited by treatment with NF340, a P2Y11 receptor antagonist. Immunofluorescent confocal imaging and western blotting analysis consistently demonstrated the P2Y11 receptor expression in Huh-7 and HepG2 cells. Transfection with P2Y11-specific siRNA attenuated the P2Y11 receptor protein expression level and also reduced NF546-induced increase in the [Ca2+]i. Importantly, immunohistochemistry revealed that the P2Y11 receptor was expressed at very high level in human HCC tissues and, by contrast, it was barely detected in normal liver tissues. Trans-well cell migration assay demonstrated that ATP and NF546 induced concentration-dependent stimulation of Huh-7 cell migration. Treatment with NF340 prevented ATP-induced stimulation of cell migration. Taken together, our results show carcinoma-specific expression of the P2Y11 receptor and its critical role in mediating ATP-inducing Ca2+ signalling and regulating cell migration in human HCC cells.