On the path toward induced pluripotency

On the path toward induced pluripotency, investigators rely on gene expression patterns as milestones to gauge appropriate progression, and comparative analysis of publically available gene expression profiles is a common approach. Clearly, our data show that inconsistencies in the relative levels of KLF4 to OCT3/4, SOX2, and c-MYC can fundamentally confound comparative analysis. Application of monocistronic Klf4 or Klf4 leads to nearly indistinguishable reprogramming phenotypes. Thus, typical clonal isolation of fully reprogrammed iPSCs with monocistronic vectors, which is subject to phenotypic selection for appropriate factor expression levels, should be mostly unaffected. Mechanistic studies, on the other hand, are prone to transcriptional noise arising from variable factor expression between cells and among transgenes. We expect the KLF4 threshold effects observed herein to be pronounced when the relative factor stoichiometry is fixed, for polycistronic transfection or even in secondary reprogramming systems with pre-integrated transgenes (Wernig et al., 2008; Woltjen et al., 2009). As such, we must be mindful that it is difficult to make direct comparisons across distinct reprogramming systems without first defining the inherent factor stoichiometry.
Experimental Procedures
Author Contributions
Acknowledgments We thank Kazutoshi Takahashi, Masamitsu Sone, and Ren Shimamoto for their critical readings of the manuscript, and Katsunori Semi for bioinformatics advice. We also thank Chiho Sakurai, Michiko Nakamura, and Akiko Oishi for technical support; Toshiko Sato for microarray preparations; Akito Tanaka for flt3 inhibitor injections; and Kanako Asano and Kotaro Ohnishi for mouse husbandry. We appreciate the advice of Hiroaki Kii (Nikon, Japan) regarding image acquisition and analysis. We also thank Darrell N. Kotton and Gustavo Mostoslavsky (Boston University School of Medicine) for providing pHAGE-Tet-STEMCCA, Alan Bradley and Kosuke Yusa (Wellcome Trust Sanger Institute) for providing pPB-CAG.OSKMBpuΔtk, Hitoshi Niwa (RIKEN Center for Developmental Biology) for providing the pLefty1-luc reporter, and all Addgene contributors, especially Linzhao Cheng and Rudolf Jaenisch. This work was supported by the Cabinet Office, Government of Japan and the Japan Society for the Promotion of Science (JSPS) through the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program), the Core Center for iPS Cell Research, the Research Center Network for Realization of Regenerative Medicine, and the Strategic International Collaborative Research Program of the Japan Science and Technology Agency (JST). K.W. is a Hakubi Center Special Project Researcher. S.Y. is a non-salaried scientific advisor of iPS Academia Japan. S.L. received funding from EPASI (JSPS/National Science Foundation, 2013). S.-I.K. is a JSPS Fellowship recipient (2011-2013) and JST Researcher.
Introduction Epiblast stem cells (EpiSCs) are pluripotent stem cells (PSCs) derived from the epiblasts of early postimplantation mouse embryos (Brons et al., 2007; Tesar et al., 2007). EpiSCs can proliferate indefinitely in culture and differentiate into derivatives of all three germ layers in vitro and in teratomas. However, EpiSCs possess several different characteristics compared with other PSCs such as mouse embryonic stem cells (mESCs). Whereas mESCs show dome-shaped colonies, EpiSCs show a flatter colony morphology. Other characteristics that differ between EpiSCs and mESCs are the status of X chromosome inactivation in female cells, the culture conditions needed, the expression of several genes/markers (e.g., mESC-specific Pecam1 and EpiSC-specific Fgf5), and their clonogenicity and contribution to chimeras when injected into blastocysts (Brons et al., 2007; Nichols and Smith, 2009). Most of these cellular characteristics of EpiSCs are shared by human PSCs such as human ESCs (hESCs) and human induced PSCs (hiPSCs). EpiSCs and human PSCs are considered “primed” PSCs, in contrast to “naïve”-type stem cells such as mESCs (Nichols and Smith, 2009). Therefore, comparisons between EpiSCs and hESCs/hiPSCs should contribute to our understanding of the nature of the primed state and provide insights into the processes underlying the naive-to-primed state transition.