EOS lentiviral vector selection system for human induced pluripotent stem cells.
Generation of induced pluripotent stem (iPS) cells from patients has exciting applications for studying molecular mechanisms of diseases, screening drugs and ultimately for use in cell therapies. However, the low efficiency and heterogeneous nature of reprogramming is a major impediment to the generation of personalized iPS cell lines. We reported in Nature Methods (6, 370-376, 2009) the first selection system to enrich for reprogrammed human iPS cells. Using a lentiviral vector that specifically expresses the enhanced green fluorescence protein and puromycin resistance genes in pluripotent stem cells, it is now possible to mark and enrich for human iPS cell colonies expressing endogenous pluripotency markers. In this study, we describe a detailed protocol for the production of the pluripotent state-specific lentiviral vector and the selection system for the induction of healthy and disease-specific human iPS cells. Overall, preparation of the selection system takes 2 weeks, and the generation of human iPS cells takes approximately 2 months.
Hotta
Akitsu
A
Developmental and Stem Cell Biology Program, SickKids, Toronto, Ontario, Canada.
Cheung
Aaron Y L
AY
Farra
Natalie
N
Garcha
Kamal
K
Chang
Wing Y
WY
Pasceri
Peter
P
Stanford
William L
WL
Ellis
James
J
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D004351:Drug Resistance / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000821:virology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000821:virology; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D014162:Transfection / Q000379:methods
D015415:Biomarkers; D049452:Green Fluorescent Proteins