Reprogramming Mouse and Human Fibroblasts to iPSCs

MEFs were seeded in DMEM, 15% FBS, penicillin-streptomycin, glutamax, β-mercaptoethanol, sodium-pyruvate, non-essential amino acids, LIF on gelatinized (0.1%) 6-well dishes at a density of 1.25×10⁵cells/10cm². After 24hrs culture, FugeneHD (Roche) was used to transfect cells with 10ng, 100ng, or 400ng of each mFx transposon (25ng, 50ng, or 100ng for PB-TET-MKOS) plus 100ng of pCyL43 PB transposase plasmid^{11 (link)} (normalized to 2μg total DNA with empty pBluescriptKS+) at a Fugene:DNA ratio of 8uL:2μg. After 24 hours, the media was supplemented with dox (d0), and changed entirely 48hrs post-transfection. Cells were fed daily with dox containing media (1.5μg/mL, unless otherwise indicated). Colonies were picked in 96-well format over d10-14 and cultivated on mitomycin-c arrested MEFs. For PB-TET induced clones, dox treatment was maintained until d16-24. iPS cells for DNA or RNA preparation were grown on gelatin. Established iPS cells were passaged 1:6 every 48 hours. Transfection of HEFs was performed similarly, except fibroblasts were initially seeded in DMEM supplemented with 15% human serum, 10ng/mL bFGF, penicillin-streptomycin, glutamax, non-essential amino acids at a density of 6.25×10⁴cells/10cm², and grown in HEScGRO (Millipore) 48 hours after transfection. Doxycycline (1.5μg/ml) was added 24h post transfection and withdrawn a week after picking. Colonies were initially passaged mechanically 1:2, and later with TripLE Select (Invitrogen) 1:4 every 7 days. Human iPS cells were maintained on inactivated MEFs in KO-DMEM, 20% serum replacement, 10ng/mL bFGF, penicillin-streptomycin, glutamax, non-essential amino acids.