Figure 3

Impact of the E181K rhodopsin gene mutation on rod photoreceptor cells derived from iPSC lines. (A) Protocol of rod photoreceptor cell differentiation. (B) Expression of the rod photoreceptor cell-specific gene Nrl was visualized by infection with the Ad-pNrl-EGFP virus. (C) Flow cytometry analysis of the differentiated cells without (left) and with (right) Ad-pNrl-EGFP infection at 5 weeks. (D) The recoverin mRNA levels of the flow cytometry-purified pNrl-EGFP-positive rod photoreceptor cells derived from the #5 iPSCs compared to the undifferentiated #5 iPSCs at the same time point. (E) Quantification of the pNrl-EGFP-positive rod photoreceptor cells in each iPSC line after 2, 3, 4, and 5 weeks of differentiation. Red, B7; blue, #5Rw; pink, B7Rm; green, #5. N = 9. (F) Proportion of pNrl-EGFP-positive rod photoreceptor cells derived from iPSC lines after 5 weeks. N = 9. (G, H) The ratio of dead cells in pNrl-EGFP-positive photoreceptor cells detected by Annexin V (G) and PI (H). N = 4. (I-L) Relative mRNA levels of BiP (I), CHOP (J), BID (K), and NOXA (L) normalized to b-Actin in the pNrl-EGFP-positive cells collected after 5 weeks, as determined by a real-time PCR analysis. N = 3. Ad-pNrl-EGFP, adenovirus promoter Nrl-EGFP. *p < 0.05. Scale bar, 40 μm. Mean ± SD (with each p-values of marked by *) for iPSCs and pNrl-EGFP cells in (D) 1 ± 0.38, 13.6 ± 0.14 (p = 0.049); for #5, #5Rw, B7, B7#Rm in (F) 34.6 ± 11.7, 55.7 ± 10.0 (p = 0.035), 64.3 ± 11.3, 38.9 ± 5.8 (p = 0.013); for #5 and #5Rw in (G) 1 ± 0.18, 0.16 ± 0.02, (p = 0.019) (H) 1 ± 0.24, 0.24 ± 0.07 (p = 0.021); for #5, #5Rw, B7, B7#Rm in (I) 1 ± 0.005, 0.83 ± 0.002 (p < 0.0001), 0.95 ± 0.006, 1.33 ± 0.004 (p < 0.0001); (J) 1 ± 0.002, 0.76 ± 0.007 (p < 0.0001), 0.79 ± 0.005, 1.03 ± 0.010 (p < 0.0001); (K) 1 ± 0.016, 0.88 ± 0.006 (p = 0.003), 0.94 ± 0.008, 1.14 ± 0.013 (p < 0.0001); (L) 1. ± 0.005, 0.75 ± 0.005 (p < 0.0001), 0.77 ± 0.005, 0.80 ± 0.004 (p = 0.001). All statistical analyses in this figure were carried out by Student’s T test.