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

Abstract

Objectives: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Müller cells; Trim9; glaucoma; retinal ganglion cells; retinal stem cells.

图1

大鼠视网膜Müller细胞的鉴定及诱导去分化 Figure 1 Identification and dedifferentiation induction of rat retinal Müller cells A: Immunofluorescence was used to detect the expression of glutamate/aspartate transporter (GLAST) and glutamate synthase (GS) in the cells. Scale bar: 50 μm. B: PCR assay was performed to detect the expression of GLAST, rhodopsin, protein kinase C (PKC), GS, Calbindin, Nestin, Thy1.1, Pax6, hematopoietic progenitor cell (HPC)-1 and Brn-3b in the cells. C: PCR assay was performed to detect the expression of Nestin and Pax6 in Müller cells treated with or without epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). D: Western blotting was performed to detect the expression of Nestin and Pax6 in Müller cells treated with or without EGF and FGF2. E: Immunofluorescence was used to detect the expression of Nestin in Müller cells treated with or without EGF and FGF2. Scale bar: 50 μm.

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荧光显微镜观察和流式细胞术评估病毒感染效率 Figure 2 Efficiency of viral infection was observed using fluoresence microscopy and assessed by flow cytometry asssay A: Fluorescence microscopy was used to observe the efficiency of viral infection. B: Flow cytometry assay was performed to test the efficiency of viral infection.

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PCR和蛋白质印迹法检测Trim9对大鼠视网膜Müller细胞定向分化为神经节细胞的调控作用 Figure 3 Regulation of Trim9 on the directional differentiation of rat retinal Müller cells into ganglion cells was detected by PCR and Western blotting A: PCR assay was used to detect the mRNA expression of Brn-3b, rhodopsin, HPC-1, protein kinase C (PKC), Calbindin, Thy1.1, and Nestin in Trim9 overexpressing retinal stem cells after brain-derived neurotrophic factor (BDNF), retinoic acid (RA) and DMEM/F12 treatment for 5, 7 or 14 days. B: Western blotting was used to test the protein expression of Brn-3b, GLAST, rhodopsin, HPC-1, PKC, Calbindin, Thy1.1, glutamine synthetase (GS), and Nestin in Trim9 overexpressing retinal stem cells after BDNF, RA and DMEM/F12 treatment for 5, 7 or 14 days. *P<0.05, **P<0.01, ***P<0.001.

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免疫荧光法检测Trim9对大鼠视网膜Müller细胞定向分化为神经节细胞的调控作用 Figure 4 Regulation of Trim9 on the directional differentiation of rat retinal Müller cells into ganglion cells was detected by immunofluorescence Immunofluorescence was performed to detect the expression of Thy1.1 and Nestin in Trim9 overexpressing retinal stem cells after brain-derived neurotrophic factor (BDNF), retinoic acid (RA), and DMEM/F12 treatment for 5, 7 or 14 days. Scale bar: 100 μm.