Pmxs nanog

The pMXs Oct4, pMXs Sox2, pMXs Klf4 and pMXs Nanog plasmids were obtained from Addgene. Dppa2, Sall4 and lin28 were amplified from a mESC cDNA library via PCR and were cloned into the pMXs vector, which resulted in the addition of an HA tag at the C terminus of the protein. Plat E cells were transfected with the pMXs vectors. The cells were then incubated overnight, and the medium was replaced with fresh medium. The virus-containing supernatants were collected 48 h after transfection and were concentrated using Retro-Concentin (SBI). Low-passage MEFs (p 1–3) were seeded 12 h prior to infection. The infections were performed in F.Gro medium, without vitamin C, that was supplemented with 4 mg/ml polybrene (Millipore) and equal amounts of each viral concentrate. After overnight incubation, the cells were washed with PBS. According to the mES protocol, 3 ml of mESC medium was added. According to the LS/2i protocol, the infected cells were maintained in F.Gro medium without vitamin C, and the medium was replaced with 2i medium 3 days after treatment. The iPSC colonies were isolated based on the expression of Oct4-GFP and ESC morphology.

The iPSC-WT cell line was derived from MRC-5 fibroblasts (ATCC), and the iPSC-DS clones were derived from AG06872 fibroblasts (Coriell). The fibroblasts were transduced with retroviral vectors (pMXs-cMyc, pMXs-Nanog, pMXs-hOct3-4, and pMXs-Sox2; Addgene) to overexpress Oct4, Sox2, Nanog, and c-Myc transgenes. The retroviral vectors were produced by transient transfection of 293T cells. Following this, the fibroblasts were incubated for 4 h in the viral supernatants containing 5 μg/mL polybrene (Sigma). The transduced cells were then incubated for 3 weeks until development of the pluripotent clones. After isolation, the clones were grown in StemPro medium (Invitrogen) on a Matrigel^® substrate (BD Biosciences). The cultures were split mechanically using the StemPro EZ Passage tool (Invitrogen).