Abstract
Purpose: :
Previous studies using the chick system provide compelling evidence for gene-directed reprogramming of RPE progeny cells to produce differentiating photoreceptors. In this study, we tested whether mammalian RPE cells can be guided by a pro-photoreceptor gene towards differentiating as photoreceptor cells.
Methods: :
Primary cell cultures were established with isolated RPE from porcine and mouse eyes. The culture was infected with a lenti virus (Lvx-IRES-ZsGreen) expressing human neurogenin1 (Lvx-ngn1-IRES-ZsGreen) or neurogenin3 (Lenti-virus Lvx-ngn3-IRES-ZsGreen1). Expression of the transgene was indirectly monitored by viewing ZsGreen1. Reprogramming of the transduced cells as differentiating photoreceptor cells were evaluated by morphology and immunostaining for photoreceptor proteins.
Results: :
Three days after viral administration, some ZsGreen1+ cells, particularly those with brighter fluorescence (likely to have higher levels of transgene expression), began to exhibit morphologies resembling differentiating photoreceptor cells. Immunochemistry showed that some ZsGreen1+ cells were positive for neural maker Map2, rhodopsin, or red opsin. Photoreceptor-like cells were detected in both P5 mouse and adult porcine RPE cell cultures.
Conclusions: :
The results indicate that RPE→ photoreceptor reprogramming can occur in primary cell cultures derived from porcine and mouse RPE.
Keywords: photoreceptors • regeneration • retinal pigment epithelium