Supplementary Figure 7: Oct4 lineage tracing and ablation systems to track the origin of iNSCs

(A) Representative images of OKSM-iNSC (EYFP⁺) colonies generated with modified NSC media after 4-5 days of doxycycline (dox) induction, followed by 17 days of dox-independent growth. Note that images depicting neurites were taken at higher magnification than those of the corresponding EYFP⁺ OKSM-iNSCs. (B) Schematic of lineage ablation approach using Oct4-CreER; R26-lsl-DTA MEFs to test whether all iNSCs pass through an Oct4⁺ stage. (C) 4-OHT treatment triggers death of Oct4⁺ cells as judged by alkaline phosphatase staining (AP) of dox-independent iPSC colonies in ESC media. The presence of residual AP⁺ colonies in 4-OHT treated Oct4-CreER; R26-lsl-DTA cells likely represents partially reprogrammed iPSCs or iPSC colonies that failed to recombine the R26-lsl-DTA allele. (D) Quantification of AP⁺, dox-independent iPSC formation shown in (C). For each replicate, 3x10⁴ cells were used (n=3 independent replicates; error bars, s.e.m.).