Trim9 regulates the directional differentiation of retinal Müller cells to retinal ganglion cells.

Abstract

Glaucoma is a leading cause of irreversible blindness, and effective therapies to reverse the visual system damage caused by glaucoma are still lacking. Recently, the stem cell therapy enable the repair and regeneration of the damaged retinal neurons, but challenges regarding the source of stem cells remain. This study aims to investigate a protocol that allows the dedifferentiation of Müller cells into retinal stem cells, following by directed differentiation into retinal ganglion cells with high efficiency, and to provide a new method of cellular acquisition for retinal stem cells. Epidermal cell growth factor and fibroblast growth factor 2 were used to induce the dedifferentiation of rat retinal Müller cells into retinal neural stem cells. Retinal stem cells derived from Müller cells were infected with a Trim9 overexpression lentiviral vector (PGC-FU-Trim9-GFP), and the efficiency of viral infection was assessed by fluorescence microscopy and flow cytometry. Retinoic acid and brain-derived neurotrophic factor treatments were used to induce the differentiation of the retinal stem cells into neurons and glial cells with or without the overexpression of Trim9. The expressions of each cellular marker (GLAST, GS, rhodopsin, PKC, HPC-1, Calbindin, Thy1.1, Brn-3b, Nestin, Pax6) were detected by immunofluorescence, PCR/real-time RT-PCR or Western blotting. Rat retinal Müller cells expressed neural stem cells markers (Nestin and Pax6) with the treatment of epidermal cell growth factor and fibroblast growth factor 2. The Thy1.1 positive cell rate of retinal stem cells overexpressing Trim9 was significantly increased, indicating their directional differentiation into retinal ganglion cells after treatment with retinoic acid and brain-derived neurotrophic factor. In this study, rat retinal Müller cells are dedifferentiated into retinal stem cells successfully, and Trim9 promotes the directional differentiation from retinal stem cells to retinal ganglion cells effectively.