Fig. 2

Transition from high to low-efficiency reprogramming during early differentiation. a Cells differentiated for our selected time intervals were seeded in serum/LIF + dox condition for the specified durations prior to dox withdrawal. The x-axis represents the dox duration and y-axis shows the normalized fraction of Nanog+ colonies after spending the rest of the 96 h experimental duration in FBS/LIF. Each colored line shows the fraction of cells for each pulse duration that were differentiated for the respective intervals. Error bars represent 25^th−75^th percentile. All experiments were performed with n = 3 biological replicates, comprised of four technical replicates. b Nanog reporter activity measured as the number of GFP⁺ pixels (log₂-transformed) per imaged field for continuously tracked undifferentiated iPSCs (0h) and cells differentiated for 48 h. Undifferentiated iPSCs expand clonally, with a constant GFP signal doubling rate of 17.3 h, while the majority of cells differentiated for 48 h begin in a Nanog::GFP^– state and switch to a Nanog::GFP⁺ state between 24 h and 36 h. During this window, the GFP⁺ area increases rapidly, with a doubling rate of 0.8 h, after which signal increases in a log-linear fashion with a doubling rate of 23.8 h. All experiments were performed with n = 3 biological replicates, comprised of four technical replicates. c For each differentiation timepoint, we performed retrospective analysis of lineages (defined as a colony formed from a re-seeded cell) from our live imaging experiments and assigned them to one of four fate outcomes: (1) Nanog::GFP⁺ lineages activate and propagate the mono-allelic GFP reporter uniformly in all subsequent cells (green bar); (2) Nanog::GFP⁺ mixed lineages generate heterogeneous colonies with stable propagation of Nanog::GFP signal and stain uniformly for NANOG antibody (ab) (yellow bar); (3) Nanog::GFP^–/ab⁺ lineages exhibit gross cellular features of an iPSC colony without activating the endogenous Nanog::GFP reporter, but stain uniformly for NANOG (orange bar); (4) Nanog::GFP^–/ab^– lineages fail to activate the reporter, do not resemble iPSC colonies, and show no signal in the NANOG antibody staining (red bar). Representative images of the four fate outcomes are shown. Scale bar = 50 µm. All experiments were performed with n = 3 biological replicates, comprised of  four technical replicates. d Quantification of the four fate outcomes (same color coding as panel c) for –dox and +dox conditions demonstrate a sharp transition from high- to low-efficiency reprogramming responses over differentiation time that is extended for ~24 h by ectopic OSKM