PLAT-A packaging cells were plated onto six well plates coated with Poly-D-Lysine at a density of 1.5×106 cells per well without antibiotics and incubated overnight. Cells were transfected with 4 μg of moloney murine leukemia-based retroviral vectors (pMXs) containing the human cDNA of POU5F1, SOX2, KLF4 or MYC (Addgene) using Lipofectamine 2000 (Life Technologies, Inc.) according to the manufacturer’s instructions. Viral supernatants were collected at 48 and 72 hours post-transfection, filtered through a 0.45 μm pore-size filter. 150,000 HDFs were seeded onto each well of a six well plate overnight. Equal volumes of fresh 48 hour and 72 hour viral polybrene-supplemented (6 μg/ml, Sigma) supernatants were added onto the cells at 24 hours and 48 hours post-seeding. On day three, the transduced cells were split onto MEFs at a density of 104 cells per well of a six well plate in hESC medium supplemented with 0.5 mM Valproic Acid (VPA, Stemgent). Cells were fed every other day with hESC medium + VPA for 14 days. iPSC colonies were picked three weeks post-transduction and transferred onto MEF plates.
