title: Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection.
abstract: 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 approxiamtely 10⁷ cells and cryopreserved within 6-8 weeks.
authors.affiliation: Wolfson Centre for Stem Cells, Tissue Engineering & Modelling, Centre for Biomolecular Sciences, University of Nottingham, UK.
authors.forename: Emily
authors.initials: E
authors.lastname: Dick
authors.forename: Elena
authors.initials: E
authors.lastname: Matsa
authors.forename: Lorraine E
authors.initials: LE
authors.lastname: Young
authors.forename: David
authors.initials: D
authors.lastname: Darling
authors.forename: Chris
authors.initials: C
authors.lastname: Denning
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D014776:Virus Cultivation