All of these studies categorize

All of these studies categorize MET as an initial event during reprogramming which is followed by upregulation of the pluripotency machinery later in the process. Here we observed that in the time course of reprogramming human fibroblasts to iPSCs, the MET was preceded by a six day period (D0–6), during which CCT251545 analogue decreased in size, rearranged their Actin cytoskeleton and retained a partly mesenchymal phenotype. Epithelial characteristics such as adherens junctions and tight junctions were not established before D12 when the pluripotency markers TRA-1-60, Nanog, UTF1 and DPPA2 were upregulated, thus suggesting a synchronized acquisition of epithelial and pluripotent traits. We speculated whether this could be due to the fibroblast culture conditions during the first six days of our reprogramming protocol, which could potentially delay MET. However no colonies emerged when we cultured our reprogramming cells in stem cell conditions from day 1 post-electroporation. Thus, we propose that the discrepancy in the timing of MET may be a matter of species-related differences in reprogramming dynamics, given that most of aforementioned studies were performed with MEFs. This is supported by three other studies of human fibroblasts undergoing reprogramming to iPSCs, which reported E-cadherin expression beginning between days 6 and 15 of reprogramming (Onder et al., 2012; Subramanyam et al., 2011b) and TRA-1-60 positive hiPSC colonies emerging between days 11 and 15 of reprogramming (Takahashi et al., 2014). It was recently shown that promoting a brief mesenchymal phase prior to the MET could enhance the formation of both mouse and human iPSC colonies (Liu et al., 2013; Di et al., 2014). The hypothesis that mesenchymal cells must undergo an EMT-like reinforcement before turning their fate towards becoming a pluripotent epithelium correlates with our preliminary observations of Beta-catenin accumulating in the nucleus during the Initiation phase of reprogramming. Nuclear translocation of Beta-catenin is a hallmark of Canonical Wnt signaling and EMT, whereas recruitment of Beta-catenin to the plasma membrane indicates the formation of epithelial adherens junctions and MET (Zeisberg and Neilson, 2009; Kam and Quaranta, 2009). Thus, the presumed movement of Beta-catenin from the cytoplasm to the nucleus between days and 6 in our reprogramming experiments, and from the nucleus to the plasma membrane between days 6 and 12, supports the sequential EMT–MET dynamics reported in Liu et al. (2013).
Conclusion Here, we provide the first combined ultrastructural, immunocytochemical and gene expression analysis of human fibroblasts at various stages of episomal iPSC reprogramming, as well as the first real-time live cell visualization of MET during reprogramming. Our results indicate that the MET occurs approximately 6–12days after electroporation in synchrony with the upregulation of pluripotency markers, and resembles a reversal of the EMT which takes place during mammalian gastrulation. The following are the supplementary data related to this article.
Author contribution
Acknowledgments The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union\'s Seventh Framework ProgrammeFP7/2007-2013/under REA grant agreement n° PIAPP-GA-2012-324451. Additionally, this work was partly supported by the Danish National Advanced Technology Foundation grant number 047-2011-1, and by the U.S. Department of Agriculture award 2011-67015-30688. Imaging data were collected at the Center for Advanced Bioimaging (CAB) Denmark, University of Copenhagen. We thank PhD Mikkel Aabech Rasmussen and Professor Alexander Schulz for their scientific advice and discussion, and Dr. Christine Gilles for providing the Vimentin promoter-GFP reporter construct. Finally, we would like to express our gratitude to Professor Kjeld Møllgård and PhD Christian Beltoft Brøchner for proof reading of the manuscript.