Figure 4

Pluripotency-inducing factors sox2 , c-myc and klf4 and eye field transcriptional factor expression is sustained in the injured retinal pigmented epithelium in the presence of FGF2. (A-C) Quantitative RT-PCR analysis of sox2, c-myc and klf4 (A); eye field transcriptional factors pax6 (5a+), pax6 (5a-), six3, six6 and lhx2, rx1 and the progenitor markers ascl1and chx10 (B); and retinal pigmented epithelium (RPE)-specific markers mitf and tyr (C) at 6, 24 and 72 h PR in the 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. Significance was determined with unpaired Student’s t-test by comparing each time point with the intact RPE (no injury). Error bars represent standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001, compared with intact RPE. (D-G) Immunofluorescence analysis of c-Myc (D), Sox2 (E), Klf4 (F) and progenitor markers Pax6 and Chx10 (G) in transdifferentiated RPE. The asterisk represents the FGF2-soaked heparin bead. The scale bar in panel G represents 50 μm and applies to panels D-G. CR, ciliary regeneration; M, mesenchyme; RPE: retinal pigmented epithelium; T, transdifferentiated RPE.