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High-resolution analysis with novel cell-surface markers identifies routes to iPS cells



The generation of induced pluripotent stem (iPS) cells presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. Although several intermediate populations have been described1,2,3,4,5,6,7, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPS cells. The rapid expansion of minor reprogrammed cells in the heterogeneous population can also obscure investigation of relevant transition processes. Understanding the biological mechanisms essential for successful iPS cell generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that in mouse embryonic fibroblasts, reprogramming follows an orderly sequence of stage transitions, marked by changes in the cell-surface markers CD44 and ICAM1, and a Nanog–enhanced green fluorescent protein (Nanog–eGFP) reporter. RNA-sequencing analysis of these populations demonstrates two waves of pluripotency gene upregulation, and unexpectedly, transient upregulation of several epidermis-related genes, demonstrating that reprogramming is not simply the reversal of the normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and the improved understanding of the reprogramming process will lead to new reprogramming strategies.

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Figure 1: FACS analysis during secondary reprogramming of MEFs with CD44/ICAM1 double staining.
Figure 2: CD44/ICAM1 subpopulations represent distinct stages of reprogramming.
Figure 3: Global gene expression changes during the stage transition.

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Data deposits

RNA-sequencing data are deposited in the ArrayExpress under accession number E-MTAB-1654.


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We thank A. Nagy and K. Woltjen for providing the 6c iPS cell line, I. Chambers for providing TNG mice, S. Monard and O. Rodrigues for assistance with flow cytometry, and T. Kunath, T. Burdon, S. Lowell and N. Festuccia for discussions and comments on the manuscript. We also thank L. Robertson for technical assistance, and Biomed unit staff for mouse husbandry. This work was supported by ERC grants ROADTOIPS (no. 261075) and BRAINCELL (no. 261063), and the Anne Rowling Regenerative Neurology Clinic. J.O.’M. and T.R. are funded by an MRC PhD Studentship and a Darwin Trust of Edinburgh Scholarship, respectively.

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Authors and Affiliations



J.O.’M. designed and performed flow cytometry analysis and sorting experiments, prepared RNA for sequencing, carried out immunofluorescence imaging, and collected, analysed and interpreted data, and wrote the manuscript. S.S. analysed RNA-sequencing and published microarray data sets. K.A.I. carried out single-cell PCR analysis. E.C. performed primary reprogramming and FACS analysis. T.R. carried out immunofluorescence and confocal imaging. S.R.T. performed microarray analysis to identify cell-surface marker candidates. A.J. and S.L. performed multiplexed RNA-sequencing and collected data. K.K. conceived the study, identified the surface markers, generated the D6s4B5 iPS cell line, analysed RNA-sequencing data, supervised experiment design and data interpretation, and wrote the manuscript.

Corresponding author

Correspondence to Keisuke Kaji.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-18. (PDF 3715 kb)

Supplementary Data

This zipped file contains Supplementary Tables 1-9. Supplementary Table 1 shows differentially expressed genes (DEGs) between samples, Supplementary Table 2 shows genes in A-E category from O'Malley subpopulation data, Supplementary Table 3 displays gene ontology from O'Malley subpopulation data, Supplementary Table 4 contains signal values of pluripotency and epidermis genes, Supplementary Table 5 shows epidermis genes EST profile, Supplementary Table 6 shows genes in pA-pD’ category from piPSC data, Supplementary Table 7 contains genes in tA’-tD category from time course data, Supplementary Table 8 shows genes in TSO A1-TSO E category from Thy1/SSEA1/Oct4-GFP subpopulation data and Supplementary Table 9 shows flow cytometry conditions and TaqMan Gene Expression Assay ID. (ZIP 3076 kb)

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O’Malley, J., Skylaki, S., Iwabuchi, K. et al. High-resolution analysis with novel cell-surface markers identifies routes to iPS cells. Nature 499, 88–91 (2013).

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