Journal
NATURE PROTOCOLS
Volume 6, Issue 6, Pages 701-714Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2011.320
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Funding
- Medical Research Council
- Biotechnology and Biological Sciences Research Council
- British Heart Foundation
- BBSRC [BB/E006159/1] Funding Source: UKRI
- MRC [G0801098, G113/30] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BBS/B/06164, BB/E006159/1] Funding Source: researchfish
- British Heart Foundation [PG/09/027/27141] Funding Source: researchfish
- Medical Research Council [G0801098, G113/30] Funding Source: researchfish
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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 hiPSC 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 hiPSC and enable lines to be isolated, expanded to similar to 10(7) cells and cryopreserved within 6-8 weeks.
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