We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.
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We thank R. Flannery for help with mouse husbandry and D. Fu, K. Saha, A.W. Cheng, F. Soldner, and M. Dawlaty for excellent assistance and helpful comments. We also thank K. Hochedlinger and M. Stadtfeld for the Col1a1-OKSM mice and for iPSC DNA and for comments on the manuscript. D.A.F. was supported by a NSF graduate research fellowship (NSF GFRP). R.J. is supported by grants from the NIH: 5-RO1-HDO45022, 5-R37-CA084198, and 5-RO1-CA087869. R.J. is an adviser to Stemgent and cofounder of Fate Therapeutics.