Supplementary Figure 15: Oct4 lineage tracing during induced cardiomyocyte (iCM) or induced neuron (iN) generation from MEFs

(A) Quantification of EYFP⁺ and Cardiac Troponin (cTnT)⁺ induced cardiomyocytes derived from Oct4-CreER; R26-lsl-EYFP MEFs upon brief OKSM expression. For each replicate, 5x10⁵ cells were used (n=3 biological replicates; error bars, s.e.m. ** P<0.005 n.s., not significant P=0.07). (B) Representative immunofluorescence images showing co-staining for EYFP and cTnT expression in induced cardiomyocytes. (C) Schematic of the lineage tracing approach to test if iN-like cells derived from Oct4-CreER; R26-lsl-EYFP MEFs upon transduction with tetOP-Ascl1, tetOP-Brn2 and tetOP-Myt1l vectors pass through an Oct4⁺ stage. (D) Lack of EYFP expression in non-transduced (upper panel) or transduced (middle panel) MEFs grown in iN transdifferentiation medium for 12 days. Tuj1⁺ iN-like cells were detected exclusively in cultures transduced with tetOP-Ascl1, tetOP-Brn2 and tetOP-Myt1l vectors. We calculated the frequency of iN formation by counting Tuj1⁺ cells per DAPI⁺ cells in 8 random fields, indicating a reprogramming efficiency of 9%. (13 iN cells/142 DAPI⁺ cells) (E) Flow cytometry analysis of bulk cultures undergoing transdifferentiation. Note the lack of EYFP expression in transduced MEFs relative to positive control (EYFP⁺ OKSM-iNSC clone #2). The PE-Cy7 channel was used to control for autofluorescence.