Method Article
Reprogramming of mouse fibroblasts into iPSCs
https://doi.org/10.1038/protex.2015.086
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This protocol is derived from the original method to generate induced pluripotent stem cells (iPSCs)1, 2, and describes the reprogramming of mouse embryonic fibroblasts (MEFs) and mouse tail tip fibroblasts (TTFs), using pMXs-based retrovirus encoding Oct4, Sox2, Klf4, and c-Myc.
MEF/TTF medium
DMEM (Gibco; 10569-010)
10% FBS (Gibco; 16000-044)
2 mM Glutamax (Gibco; 35050-061)
0.1 mM 2-mercaptoethanol (Gibco; 21985-023)
100 units/ml penicillin and 100 µg/ml streptomycin (Gibco; 15140-122)
Plat-E medium
DMEM (Gibco; 10569-010)
10% FBS (Gibco; 16000-044)
2 mM Glutamax (Gibco; 35050-061)
0.1 mM 2-mercaptoethanol (Gibco; 21985-023)
ESC medium
Knockout DMEM (Gibco; 10829-018)
10% FBS (Gibco; 16141-079)
10% KSR (Gibco; 10828-028)
2 mM Glutamax (Gibco; 35050-061)
0.1 mM nonessential amino acids (Gibco; 11140-050)
0.1 mM 2-mercaptoethanol (Gibco; 21985-023)
103 units/ml leukemia inhibitory factor (Stemgent; 03-0011-100)
100 units/ml penicillin and 100 µg/ml streptomycin (Gibco; 15140-122)
DPBS (Gibco; 14190-144)
0.25% Trypsin-EDTA (Gibco; 25200-056)
FuGENE HD Transfection Reagent (Promega; E2311)
Opti-MEM® I Reduced Serum Medium (Gibco; 31985-047)
Preparation of MEFs
Preparation of TTFs
Reprogramming of fibroblasts
Maintain Plat-E packaging cells in Plat-E medium.
Split cells into 6-well plates at a density of 8x106 cells per plate.
On the next day, transfect Plat-E cells with pMXs plasmids encoding Oct4, Sox2, Klf4 and cMyc individually, using FuGENE HD, according to manufacturer's instruction. For each well, dilute 3 μg plasmid and 9 μl FuGENE HD transfection reagent in 200 μl Opti-MEM I Reduced Serum Medium. The DNA/FuGENE HD mixtures are incubated for 30 min at room temperature, and added onto Plat-E cells.
Change fresh Plat-E medium 1 day after transfection.
After an incubation for 24 h, collect virus-containing supernatants, supplement with polybrene (1:2000 dilution), and filter the mixtures through 0.45 μM cellulose acetate filters.
Combine and mix Oct4, Sox2, Klf4 and c-Myc viruses with equal volume.
Incubate MEFs (at passage 2 or 3) or TTFs (at passage 1 or 2) that are cultured on 0.1% gelatin-coated plates with virus/polybrene-containing supernatants for 24 h.
Change ESC medium after transduction.
Continuously culture reprogramming cells with ESC medium for at least 2 weeks.
We thank all Ding lab members for their help, and Gary Howard for editing the protocol.
The authors declare no conflicting financial interests.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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