A slight modification of the manufacturer's instructions is used — please refer to the user manual. This protocol includes the use of reprogramming enhancing chemicals which may be unnecessary and could be left out. Alternatively, the number of infected cells plated on MEFs may be reduced.
- Day -1 to -3 Plate target fibroblasts on 1 well of a 6-well tissue culture plate
- Day 0 When cells are at ~80-90% confluence add Sendai virus cocktail according to the manufacturer's instructions in 1ml fibroblast medium (10% FBS in DMEM)
- Day 1 Change medium to fibroblast medium containing reprogramming chemicals — 5uM PS48, Reagents Direct; 0.5uM A-83-01, StemGent; 0.25mM Sodium Butyrate, StemGent
- Day 3 Feed as above
- Day 5 Feed as above
- Day 6 Prepare a 10cm plate of irradiated MEF feeders according to the manufacturer's instructions — 1.5 x 106 cells
- Day 7 Dissociate the fibroblasts with Accutase™ or Trypsin and count. Resuspend cells in fibroblast medium containing reprogramming chemicals and 5uM Y27632. Plate 1 x 106 cells on the 10cm dish of irradiated feeders and retain remaining cells for RNA extraction.
- Day 8 to Day 11–12 Feed daily with hESC medium (20% Knockout Serum Replacer in DMEM/F12 + 4ng/ml bFGF) containing reprogramming chemicals, but not Y27632, until colonies start to appear.
- Day 11–12 to Day 14 Feed daily with hESC medium without chemicals
- Day 15 to Day 21–28 Feed daily with MEF-conditioned medium + 10ng/ml bFGF
- Day 21–28 Pick colonies into individual wells of 24-well plates of MEFs or Matrigel
N.B. The remaining colonies may be detached with collagenase in bulk and replated on Matrigel to allow further isolation of colonies.