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Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency

Abstract

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome–specific mouse and human iPS cell lines with known mutations in MECP2.

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Figure 1: EOS lentiviral vectors in mouse ES cells.
Figure 2: EOS lentiviral vectors in human ES cells.
Figure 3: EOS lentiviral vector in reprogrammed mouse iPS cells.
Figure 4: EOS lentiviral vector in reprogrammed human iPS cells.
Figure 5: EOS lentiviral vector in Rett syndrome–specific mouse and human iPS cell lines.

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Acknowledgements

We thank H.R. Schöler (Max Planck Institute for Molecular Biomedicine) for providing Oct-4 promoter plasmid, T. Kitamura (Institute of Medical Science, University of Tokyo) for Plat-E cells, B. Alman and A. Lin for human fibroblast isolation and I.H. Park for advice on human iPS cell isolation. We gratefully acknowledge the assistance of T. Thompson at the Ontario Human iPS Cell Facility, SickKids The Centre for Applied Genomics Facility, SickKids ES Facility, SickKids Flow Facility and the Centre for Modeling Human Disease pathology core. This work was supported by grants from the Canadian Institutes of Health Research (MOP-10825 to D.L.M., MOP-77803 to J.R., MOP-81129 and IG1-94505 to J.E., and RMF-92090 to J.E. and D.L.M.), the Stem Cell Network (to J.R., M.B. and J.E.), the Ontario Ministry of Research and Innovation (to J.R. and J.E. for the Ontario Human IPS Cell Facility), and the International Rett Syndrome Foundation (to J.E.). A.H. is supported by a Restracomp Award from SickKids Hospital, A.Y.L.C. by a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada, N.F. by an Ontario Council of Graduate Studies Master's Autism Scholars Award and the Ontario Student Opportunity Trust Funds Hayden Hantho Award and C.A.S. by the Stem Cell Network and the Juvenile Diabetes Research Foundation.

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Authors and Affiliations

Authors

Contributions

A.H., D.L.M. and J.E. conceived the project; I.A.M. provided reagents; A.H., M.B., J.R. and J.E. designed experiments; A.H. developed the EOS vectors and performed the iPS cell reprogramming experiments; A.Y.L.C. and N.F. performed the Rett syndrome iPS cell experiments; K.V. and J.S.D. performed the hES cell experiments; C.A.S. and P.P. performed the teratoma experiments; A.H., J.R., M.B. and J.E. wrote the manuscript.

Corresponding author

Correspondence to James Ellis.

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Supplementary Figures 1–12, Supplementary Tables 1 and 2 (PDF 4397 kb)

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Hotta, A., Cheung, A., Farra, N. et al. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency. Nat Methods 6, 370–376 (2009). https://doi.org/10.1038/nmeth.1325

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