Reprogramming retinal pigment epithelium to differentiate toward retinal neurons with Sox2

Abstract

Guiding non-neural, retinal pigment epithelium (RPE) to produce retinal neurons may offer a source of developing neurons for cell-replacement. Sox2 plays important roles in maintaining neural progenitor/stem cell properties and in converting fibroblasts into pluripotent stem cells. This study tests the possibility of using Sox2 to reprogram RPE to differentiate toward retinal neurons in vivo and in vitro. Expression of Sox2 in the chick retina was detected in progenitor cells, in cells at a discrete location in the layers of amacrine and ganglion cells, and in Muller glia. Overexpression of Sox2 in the developing eye resulted in hypopigmentation of the RPE. In the affected regions, expression of retinal ganglion cell markers became apparent in the RPE layer. In RPE cell culture, Sox2 promoted the expression of retinal ganglion and amacrine markers, and suppressed the expression of genes associated with RPE properties. Mechanistic investigation using the developing retina revealed a coexpression of Sox2 and basic fibroblast growth factor (bFGF), a growth factor commonly used in stem cell culture and capable of inducing RPE-to-retina transdifferentiation (or reprogramming) during early development. Similar patterns of changes in Sox2 expression and in bFGF expression were observed in atrophic retina and in injured retina. In RPE cell culture, Sox2 and bFGF mutually enhanced one another's expression. Upregulation of bFGF expression by Sox2 also occurred in the retina. These results suggest that Sox2 can initiate a reprogramming of RPE cells to differentiate toward retinal neurons and may engage bFGF during the process.

Fig. 1

Spatial and temporal pattern of Sox2 expression in chick retina. The in situ hybridization signals were visualized with nitroblue tetrazolium (A-D), fluorescein-tyramide (E,F), or rhodamine-tyramide (G,H). A: E5 retina. B: E7 peripheral retina. C: E7 central retina. D: E7 central retina at higher magnification. E: E9 retina; F: E15 retina. G: P0 retina. H: P30 retina. I-K: Double-labeling for Sox2 expression and for Muller glia protein vimentin (recognized by Mab H5) in E15 retina. I: In situ hybridization detection of Sox2 mRNA with rhodamine-tyramide. J: Immunostaining for vimentin with fluorescein. K: Simultaneous view of both stainings. Arrows in D: positive cells in the inner plexiform layer. Arrows in E and F: positive cells that appear spatially paired. Arrows in I-K point to double-labeled cells. AM: amacrine cells. GCL: ganglion cell layer. L: lens. MG: Muller glia. NE: neuroepithelium. ONL: outer nuclear layer. Scale bars: 50 μm.

Fig.2

RPE de-pigmentation from Sox2 overexpression. A: E15 eyes from an embryo infected with RCAS-Sox2 (left) or RCAS-GFP (right). The “patchy” area is encircled by a yellow line. B: Immunostaining with anti-viral protein P27 to detect viral infection in E15 retina infected with RCAS-GFP. C: Immuno-detection of viral infection (anti-viral protein P27 staining) in E15 retina infected with RCAS-Sox2. D: Immuno-detection of viral infection (with anti-viral protein P27) in E15 retina infected with RCAS-GFP. Blue lines mark regions with de-pigmentation of the RPE and retinal detachment. E-F: BrdU incorporation in de-pigmented regions of E15 retina infected with RCAS-Sox2. E: A bright-field views. F: BrdU immunostaining for BrdU. The arrows show BrdU⁺ cells in the RPE layer. GCL: ganglion cell layer. INL: inner nuclear layer. ONL: outer nuclear layer. Scale bars: 50 μm.

Fig. 3

Ectopic expression of retinal ganglion cell markers in the RPE of E18 embryos infected with RCAS-Sox2. A: Immunostaining (in red) with RA4. B: Higher magnification of the left side of A. C: Higher magnification of the right side of A. D: Immunostaining (FITC) with 3A10 of retina infected with RCAS-Sox2. E: Nuclear staining of C. F: Immunostaining (FITC) with 3A10 of retina infected with RCAS-GFP. G: Immunostaining (FITC) with 4H6 of retina infected with RCAS-Sox2. H: Nuclear staining of C. I: Immunostaining (FITC) with 4H6 of retina infected with RCAS-GFP. Arrows point to cells with neuron-like morphologies. Arrowheads point to immunostaining of the neural fibers of retinal ganglion cells. GCL: ganglion cell layer. INL: inner nuclear layer. ONL: outer nuclear layer. Scale bars: 50 μm.

Fig. 4

Suppression of RPE properties and induction of neural genes by Sox2 in RPE cell cultures. A: A schematic diagram showing the structural regions included in each recombinant DNA construct. B: Appearance of RPE cell cultures, showing hypopigmentation of the infected with RCAS-Sox2 as compared with the control infected with RCAS-GFP. Also shown are parallel cultures infected with RCAS expressing Sox2ΔB, Sox2ΔC, or En-Sox2. C-J: Expression of retinal neural markers in RPE cell cultures infected with RCAS-Sox2. C-E: Immunocytochemistry with RA4 of cultures infected with RCAS-GFP, RCAS-Sox2, and RCAS expressing 3 mutations constructs of Sox2: Sox2ΔB, Sox2ΔC, and En-Sox2. F-H: Immunocytochemistry with 3A10. I, J: Immunocytochemistry for AP2α. The arrow in the inset of D: a cell displaying morphology typical of a retinal ganglion cell. K: RT-PCR analysis of the expression of RPE genes (Mitf, Otx2, and Mmp115) and of neural genes (Npy-1 and Pax6) in RPE cell cultures infected with RCAS-Sox2 or RCAS-GFP. Small ribosomal protein 17 (S17) was used as an internal control for the amount of first-strand cDNA present in the samples. Scale bars: 50 μm.

Fig.5

The spatial pattern of bFGF expression in the developing chick retina and its co-expression with Sox2. A-D: In situ hybridization for bFGF expression in E6 (A), E8 (B), E10 (C), and E12 (D) chick retina. E-F: Double in situ hybridization for Sox2 mRNA (E, fluorescein-tyramide, in green) and bFGF mRNA (F, rhodamine-tyramide, in red) in E8 chick retina. G: A merged view of E and F. H: Hoechst nuclear staining. Arrows point to double-labeled cells in the neuroepithelial layer (NE). AM: amacrine cells. GCL: ganglion cell layer. MG: Muller glia. NE: neuroepithelium. ONL: outer nuclear layer. Scale bars: 50 um.

Fig. 6

Similar patterns of changes in expression of Sox2 and bFGF in Muller glia atrophic retina and after chemically induced retinal injury. A-F: E10 retinas partially infected with RCAS-NSCL2, which causes Muller glia atrophy [ref]. A, D: Immunostaining for antiviral protein p27 to identify regions infected by RCAS-NSCL2. B, E: Immunostaining for Muller glial protein vimentin. C, F: In situ hybridization for bFGF expression (C) and Sox2 expression (F). Arrows point to RCAS-NSCL2 infected regions with fewer vimentin⁺ cells and fewer bFGF⁺ cells or with fewer Sox2⁺ cells than the adjacent, uninfected regions. G: RT-PCR analysis of Sox2 and bFGF expression in control and in NMDA-damaged retina. H: A plot of IOD ratios to S17 of the DNA band intensities of Sox2 and bFGF PCR products. Shown are the means and S.D.s from 3 independent RT-PCR reactions. “*” indicates statistically significant at p<0.05 level, and “**” at p<0.01 level. Scale bars: 50 um.

Fig. 7

Increasing bFGF expression and decreasing PEDF expression by Sox2. A: RT-PCR analysis of bFGF and PEDF expression in E6 retina infected with RCAS-GFP (GFP) or RCAS-Sox2 (Sox2). Housekeeping gene S17 was used as an internal control of the relative amount of cDNA present in each sample. B: A plot of IOD ratios (to S17) of the DNA band intensities of PEDF and bFGF PCR products. C, D: RT-PCR and a plot of IOD ratios (to S17) of PEDF expression in E6 and E15 RPE infected RCAS-GFP (GFP) or RCAS-Sox2 (Sox2). E, F: RT-PCR and a plot of IOD ratios (to S17) of bFGF expression and PEDF expression in glial cell culture infected with RCAS-GFP (GFP) or RCAS-Sox2. G, H: RT-PCR and a plot of IOD ratios (to S17) of bFGF expression E6 RPE cell culture infected with RCAS-GFP (GFP), RCAS-Sox2ΔB, RCAS-Sox2ΔC, RCAS-En-Sox2, or RCAS-Sox2. I, J: RT-PCR and a plot of IOD ratios (to S17) of the expression of PEDF and Sox2 in RPE cell cultures infected with RCAS-GFP (GFP), treated with bFGF (bFGF), infected with RCAS-Sox2 (Sox2), or treated with bFGF as well as infected with RCAS-Sox2 (bFGF Sox2). Bars shown in all plots are the means and S.D.s from 3 independent RT-PCR reactions. “*” indicates statistically significant at p<0.05 level, and “**” at p<0.01 level.