FIGURE 5.
Reprogramming of MEFs with SeVdp vectors installed with reprogramming genes. A, the genome structure of SeVdp(c-Myc/Klf4/Oct4/Sox2) is shown. B, shown is genome structure of SeVdp(Klf4/Oct4/Sox2) and SeVdp(Zeo/hKO/c-Myc), coexisting in a single cell, is shown. C and D, shown is the efficiency to reprogram MEF/Nanog-GFP. MEF/Nanog-GFP cells (1.25 × 105) were infected with SeVdp vectors, and retroviral vectors were installed with c-Myc/Klf4/Oct4/Sox2 as described under “Experimental Procedures.” Then 1.0 × 103 of infected cells were seeded onto the feeder cells in 6-well plates and cultured for 14 days (C) or for the indicated days (D). The number of iPS colonies expressing GFP was determined under fluorescent microscopy. Reprogramming efficiency was indicated as the ratio of the number of EGFP-positive colonies to that of MEF/Nanog-GFP seeded in the well (C) or to that of infected MEF/Nanog-GFP seeded in the well (D). C, shown is a comparison of reprogramming efficiency with the SeVdp(c-Myc/Klf4/Oct4/Sox2) vector and with retroviral vectors. SeVdp(M/K/O/S), SeVdp(c-Myc/Klf4/Oct4/Sox2); RvMX4, coinfection of ecotropic retroviral vectors installed with c-Myc, Klf4, Oct4, and Sox2 separately. D, shown is a comparison of reprogramming efficiency by a single infection of SeVdp(c-Myc/Klf4/Oct4/Sox2) (SeVdp(M/K/O/S)) and by coinfections of SeVdp(Klf4/Oct4/Sox2) and SeVdp(Zeo/hKO/c-Myc) (SeVdp(K/O/S) + SeVdp(M)). E, characterization of mouse iPS cells generated with SeVdp(c-Myc/Klf4/Oct4/Sox2) is shown. The ES-like colonies emerging from MEF/Nanog-GFP cell lines were fixed, incubated with specific primary antibodies against SeV NP antigen (left), Sox2 (middle), and SSEA-1 (right), then stained with secondary antibodies conjugated with Alexa 555. The cells were then counterstained with DAPI and examined by fluorescence microscopy as described under “Experimental Procedures.” Nanog (left), expression of GFP driven by the Nanog promoter. F, gene expression analysis with semiquantitative RT-PCR is shown. Aliquots (2 μg) of total RNA prepared from the cells indicated were analyzed as described under “Experimental Procedures.” Lane 1, MEF; lane 2, MEF infected with control vector (SeVdp(Bsr/ΔF/KO)); lane 3, MEF infected with SeVdp(M/K/O/S) on day 5 infection; lane 4, SeVdp-iPS cell clone #2-1; lane 5, SeVdp-iPS cell clone #9-1; lane 6, SeVdp-iPS cell clone #13; lane 7, SeVdp-iPS cell clone #16; lane 8, SeVdp-iPS cell clone #21; lane 9, mouse iPS cell generated with retrovirus vectors (RvMX4); lane 10, mouse ES cell (clone D3). ECAT1, ES cell-associated transcript 1; FGF4, fibroblast growth factor 4. G, methylation analysis of Oct4 and Nanog promoters is shown. Methylation profile of CpG in genomic DNA was analyzed by bisulfite sequence analysis as described under “Experimental Procedures.” Open circles, unmethylated cytosine; closed circles, methylated cytosine. The ratio of methylated cytosine is indicated as a percentage of total cytosine residues analyzed. H, a telomerase assay is shown. Telomerase activity in total cell extract prepared from 1 × 103 cells was analyzed as described under “Experimental Procedures” and is indicated as the amount of (dTTAGGG)n synthesized. I, histology of teratomas derived from SeVdp-iPS cells is shown. Teratoma formation was studied at 6 weeks after the subcutaneous injection of 1 × 106 SeVdp-iPS cells from clone #13 into SCID mice. a–c, low magnification; scale bar = 100 μm. d–h, high magnification observation; scale bar = 20 μm. d, cartilage; e, epidermis; f, hair follicle; g, sweat gland; h, intestinal gland. J, adult chimeras derived from SeVdp-iPS cells (clone #13) are shown. Dark hair indicates donor contribution.