The protocols described here address methods used in two crucial stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. The first is an optimized method for producing lentivirus at an efficiency 600-fold greater than previously published, and it includes conjugation of the lentivirus to streptavidin superparamagnetic particles; this process takes 8 d. The second method enables the isolation of true hiPSCs immediately after somatic cell reprogramming and involves column-based positive selection of cells expressing the pluripotency marker TRA-1-81. This process takes 2 h and, as it is directly compatible with feeder-free culture, the time burden of manually identifying and mechanically propagating hiPSC colonies is reduced drastically. Taken together, these methods accelerate the production of hiPSCs and enable lines to be isolated, expanded to ∼107 cells and cryopreserved within 6–8 weeks.
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This work was supported by the Medical Research Council, the Biotechnology and Biological Sciences Research Council and the British Heart Foundation. We would like to thank our collaborators at the University of Newcastle International Centre for Life who provided fibroblast samples from the BioBank in Newcastle, which is part of the MRC centre for Neuromuscular Diseases and EuroBioBank.
The authors declare no competing financial interests.
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Dick, E., Matsa, E., Young, L. et al. Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection. Nat Protoc 6, 701–714 (2011). https://doi.org/10.1038/nprot.2011.320
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