Figure 5

Lin-28 is sufficient to induce retinal pigmented epithelium transdifferentiation. (A) Quantitative RT-PCR analysis at 6, 24 and 72 h post-retinectomy (PR) shows the relative levels of lin-28 expression in the injured retinal pigmented epithelium (RPE) in the absence or presence of FGF2. The expression levels were normalized with intact RPE (no injury). The analysis was performed using three independent biological samples (n = 3) in triplicate and the comparative cycle threshold (2^-ΔΔCt) method was used to determine relative changes in transcripts compared with gapdh mRNA levels. The Student’s t-test was used to determine significance. Error bars represent standard error of the mean. **P < 0.01; ***P < 0.001, compared with intact RPE. (B) Lin-28 immunofluorescence in the transdifferentiated RPE 72 h PR in presence of FGF2. (C) Magnification of the boxed area in B stained for Lin-28 (green) and DAPI (blue). (D-F) Hematoxylin-and-eosin-stained sections at 72 h PR of electroporated eyes with pcDNA3.1 + pIRES-GFP (D), pCLIN-28 + pIRES-GFP (Lin-28 + GFP) (E) or treated with FGF2 (F). (G,H) GFP immunofluorescence analysis of electroporated eyes with pIRES-GFP (G) or pCLIN-28 + pIRES-GFP (Lin-28 + GFP) (H). (I) Percentage of eyes showing transdifferentiation at 72 h PR in the presence of FGF2 (n = 12, 100%); electroporated with pCLIN-28 + pIRES-GFP (Lin-28a + GFP) (n = 33, 48%, including the thickened depigmented RPE to full RPE transdifferentiation); or pIRES-GFP (n = 17, 0%). (J) Quantitative analysis of transdifferentiated areas observed in histological sections from eyes treated with FGF2 or electroporated with pCLIN-28 + pIRES-GFP. Error bars represent standard error of the mean. The asterisk represents the FGF2-soaked heparin bead. The scale bar in panels B, E and F represents 50 μm. The scale bars in panels C, D, and G and H represent 300 μm, 100 μm, and 20 μm respectively. L: lens; M: mesenchyme; NS: non-significant; RPE: retinal pigmented epithelium; T: transdifferentiated RPE.