Research Paper Volume 8, Issue 7 pp 1330—1349

Figure 1. Mitophagy deficiency is a reprogramming barrier. (A) Early passage PINK1^+/+ and PINK1^−/− MEFs were transduced with retroviral vectors encoding for Oct4, Sox2, and Klf4 and cultured in ES medium. Phase-contrast microphotographs of representative PINK1^+/+ and PINK1^−/− MEFs at day 7 (D7) after the initial transduction with OSK are shown (white arrows indicate emerging iPSC-like colonies). Representative photographs of colonies of AP-stained OSK-transduced PINK1^+/+ and PINK1^−/− MEFs. The number of AP⁺ colonies was counted 14 days after the initial infection and represent reprogramming efficiency relative to PINK1^+/+ MEFs (x-fold) (n=6 for each condition). **, P<0.01. (B) Individual iPSC-like colonies were randomly selected from each PINK1 subtype, cultured on 6-well plates coated with MEF feeder layers, and stained either for AP activity (left) or with antibodies against Oct3/4, Nanog, Sox2, and Ssea-1 (right), as indicated in the “Methods” section. Nuclear staining was performed with Hoechst 33258. Panel depicts representative images of iPSC colonies that were captured using different channels for Oct3/4 (green), Nanog (green), Sox2 (green), Ssea-1 (green), or Hoechst 33258 (blue), as specified. Representative Western blots for Oct3/4 and Sox2 protein expression in PINK1^+/+- PINK1^−/−-iPSCs are shown (middle; n = 2).