Fibroblasts at passage 5–6 were plated at 5 × 104 cells in 35-mm tissue culture dishes in fibroblast medium and transduced with retroviral constructs expressing the pluripotency factors OCT4, SOX2, KLF4 and c-MYC.41 Fibroblasts were transduced on day 1 after plating, with daily medium changes from day 3 to day 6. On day 7, forming iPSC were trypsinized and passaged to mouse embryonic fibroblast (MEF) feeders for expansion in hESC medium compromising DMEM/F12 (Invitrogen, 11320–032), 20% KOSR (Invitrogen, 10828–028), 4 ng ml−1 FGF2 (EMD Millipore GF003), 1% NEAA, 2 mM glutamax and 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St Louis, MO, USA, M3148). After 10 days on MEFs, medium was changed to MEF-conditioned hESC medium, then hESC-like colonies manually dissected and passaged to new MEFs for expansion in hESC medium. From each fibroblast line, 5–10 colonies were selected for expansion to iPSC lines; a minimum of 5–6 iPSC lines were derived from each fibroblast (patient) sample.
