Thus, using 2Gi2R as a sensitive NCC reporter, we were able to identify an age-dependent impairment of NCC in old donor-derived human fibroblasts. Further, as 3-week-old iNs showed aging transcriptome signatures, including decreased RanBP17 levels in the old, we asked whether mature and functional iNs from old donors possessed altered NCC. We transduced iNs with the 2Gi2R reporter and let them mature on astrocytes until week 6 (Figure 6F). Mature neurons showed complex morphologies and solid expression of the NCC reporter proteins (Figure 6G). Strikingly, iNs from the oldest donors showed significantly increased GFPnuc/RFPnuc ratios compared to both middle-aged (29 year–50 year) and young donor-derived iNs (Figure 6H). Strongly affected neurons showed obvious compartmentalization defects; the extent of the detected loss of neuronal NCC significantly correlated with donor age and RanBP17 expression (Figure S6). These data show that, in addition to transcriptomic changes after 3 weeks of conversion, mature human iNs show functional NCC impairment, thus implicating NCC as an important functional signature of old human neurons. To confirm age-related NCC impairment, we next applied fluorescence loss in photobleaching (FLIP) as a second assay to measure compartmentalization to our aging fibroblasts. For this, we performed live-cell analysis of a shuttling
