Dataset: Dynamic single-cell imaging of direct reprogramming reveals an early specifying event
The study of induced pluripotency often relies on experimental approaches that average measurements across a large population of cells,...
The study of induced pluripotency often relies on experimental approaches that average measurements across a large population of cells, the majority of which do not become pluripotent. Here we used high-resolution, time-lapse imaging to trace the reprogramming process over 2 weeks from single mouse embryonic fibroblasts (MEFs) to pluripotency factor-positive colonies. This enabled us to calculate a normalized cell-of-origin reprogramming efficiency that takes into account only the initial MEFs that respond to form reprogrammed colonies rather than the larger number of final colonies. Furthermore, this retrospective analysis revealed that successfully reprogramming cells undergo a rapid shift in their proliferative rate that corresponds to a reduction in cellular area. This event occurs as early as the first cell division and with similar kinetics in all cells that form induced pluripotent stem (iPS) cell colonies. These data contribute to the theoretical modeling of reprogramming and suggest that certain parts of the reprogramming process follow defined rather than stochastic steps. Gene expression profiling: Mouse E13.5 fibroblasts were generated by blastocyst injection with doxycycline inducible Oct4, Sox2, Klf4, and c-Myc primary iPS cells as previously described. Cells were serum starved with 0.5% FBS containing medium, followed by either continued serum starvation for 96 hours, or by 96 hours of factor induction through medium supplemented with 2 ug/ml doxycycline (Sigma). Two biological replicates of each sample (SerumStarveControl and 96hrInduction) were collected and RNA was isolated in preparation for analysis on Affymetrix Mouse Genome 430 2.0 Arrays as previously described.
- Dec.12, 2014
- Nov.11, 2014