Figure 3

Dedifferentiation of retinal stem cells. The stem cells differentiated from retinal Müller cells were transfected with lentivirus PGC-FU-Atoh7-GFP (A). FACS analysis showed that the transfection efficiency was 75.41% at 48 h after transfection (B). The GFP green fluorescence could be seen perspicuously in differentiated cells (C). Double immunocytochemical analysis showed that ganglion cells expressed both Thy1.1 and Brn-3b (D). Scale bar: 100 μm. Ten different visual fields (×100) in each group were selected to count the percentage of ganglion cells. The percentage of ganglion cells was similar between Group B and Group C (13.3 ± 13.25% versus 9.10 ± 3.21%), but significantly higher in Group A (50.40 ± 8.22%) (E). At 7 and 14 days, the percentage of ganglion cells was similar (E, F). Apart from ganglion cells, retinal stem cells differentiated into other type of cells such as amacrine cells, horizontal cells, bipolar cells, photoreceptor cells and Müller cells (G-I). Scale bar: 100 μm. RT-PCR and Western blot analysis showed that the expression of Atoh7, Brn-3b and Isl-1 was increased after lentivirus PGC-FU-Atoh7-GFP transfection, but Notch1 expression was slightly decreased (J, K). Lane M: DNA marker; lane 1: groups without transfection; lane 2: groups with transfection by lentivirus PGC-FU-Atoh7-GFP. FACS, fluorescence-activated cell sorting.