Article abstract
Nature Methods 6, 370 - 376 (2009)
Published online: 26 April 2009 | doi:10.1038/nmeth.1325
Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
Akitsu Hotta1,2, Aaron Y L Cheung1,3, Natalie Farra1,3, Kausalia Vijayaragavan4,6, Cheryle A Séguin1,2,6, Jonathan S Draper1,6, Peter Pasceri1, Irina A Maksakova5, Dixie L Mager5, Janet Rossant1,2,3, Mickie Bhatia4 & James Ellis1,2,3
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.
- Developmental and Stem Cell Biology Program, Toronto, Ontario, Canada.
- Ontario Human iPS Cell Facility, SickKids, Toronto, Ontario, Canada.
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
- McMaster Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, and Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
- Terry Fox Laboratory, British Columbia Cancer Agency, and Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.
- Present addresses: Department of Regenerative Cardiology, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (K.V.), Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada (C.A.S.) and McMaster Stem Cell and Cancer Research Institute, Michael G. DeGroote Centre for Learning and Discovery, Hamilton, Ontario, Canada (J.S.D.).
Correspondence to: James Ellis1,2,3 e-mail: jellis@sickkids.ca
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