Fig. 1

A somatic-like barrier to reprogramming is established early during differentiation. a Schematic of experimental system to measure reversion back to a NANOG⁺ state by extrinsic or intrinsic conditions. At set intervals following 2i/LIF withdrawal, single cells are re-seeded into serum/LIF, either in the absence (– dox) or presence (+dox) of doxycycline (2 µg/ml) to induce ectopic OSKM expression. The number of NANOG⁺ colonies was counted after 96 h. b Representative IF images showing the loss of NANOG signal over differentiation (“diff.”, top row) and the distinct NANOG⁺ colony-forming efficiency under –dox and +dox conditions (imaged 96 h after the respective differentiation times; scale bar = 200 µm). All experiments were performed with n = 3 biological replicates, comprised of four technical replicates. c Distribution of NANOG signal (by immunostaining) within RFP-segmented colonies for differentiating cells (“diff.”, gray), cells re-seeded into serum/LIF (“– dox”, light green), or the serum/LIF with OSKM induction (“+ dox”, dark green). Distributions are over the fraction of colony area that is NANOG positive. White dot indicates the median; boxes represent interquartile range showing central 50% of data and whiskers indicate 25^th and 75^th percentile data. n > 500 for 96 h and n  > 9,000 for 24–72 h conditions. All experiments were performed with n = 3 biological replicates, comprised of four technical replicates. d Efficiency of iPSC colony-forming ability (fraction of NANOG positive pixels over the colonies) generated from IF images (panel b) and normalized to iPSC controls. NANOG⁺ colonies are computationally segmented, counted and normalized to the number of NANOG⁺ colonies generated from undifferentiated (0 h) iPSCs placed into serum/LIF. Data points represent the median values from panel c normalized to the control. Error bars represent the 25^th−75^th percentile. Grey highlighted regions indicate the 25^th−75^th percentile range for the estimated transition times (medians, vertical black lines) for the two conditions. Error bars for + dox are shifted to the right. Note that values are normalized ratios with respect to 0h, hence error bars may extend beyond 1. All experiments were performed with n = 3 biological replicates, comprised of four technical replicates. e Quantification of Alkaline Phosphatase positive colonies after 12 days of dox induction from iPSCs differentiated for 96 h or secondary MEFs generated from the same iPSC line. Reprogramming efficiency is calculated as the number of colonies per cells plated. Error bars indicate standard deviation of n = 3 reprogramming experiments in technical replicates