Characterization of iPSCs derived using the small molecule-aided feeder-free condition.

<p>(<b>A</b>) Bright-field image of iPSCs derived from human adult skin fibroblasts (iPS(SK46) clone 2). Scale bar: 100 µm. (<b>B</b>) G-banding chromosome analysis of iPS(SK46) clone 2 (p17). (<b>C</b>) PCR analysis of reprogramming vectors in iPSCs. E: episomal DNA; G: genomic DNA; NF: neonatal foreskin fibroblasts (p5); iPSF7 clone 1 to 3: iPSCs derived from neonatal foreskin fibroblasts (p26); AF: adult skin fibroblasts (p6); iPS(SK46) clone 1 to 3: iPSCs derived from adult skin fibroblasts (p22). piPSC derived from human foreskin fibroblasts (p4) were used as controls. <i>T-OCT4</i>: transgene <i>OCT4</i>; <i>T-SOX2</i>: transgene <i>SOX2</i>; <i>T-NANOG</i>: transgene <i>NANOG; T-LIN28</i>: transgene <i>LIN28</i>; <i>T-c-MYC</i>: transgene <i>c-MYC</i>; <i>T1-KLF4</i>: transgene <i>KLF4 (1)</i>; <i>T2-KLF4</i>: transgene <i>KLF4 (2)</i>; <i>T-SV40LT</i>: transgene <i>SV40LT</i>; <i>OCT4</i>: endogenous <i>OCT4</i>. 32 PCR cycles were used for all primer sets. (<b>D</b>) Quantitative RT-PCR analysis of the endogenous OCT4, NANOG, SOX2 and LIN28 expression in iPSC clones. Data shown are mean ± SEM (n = 3). (<b>E</b>) Bisulfite-sequencing analysis of the methylation status of the <i>OCT4</i> and <i>NANOG</i> promoters in iPSC clones. Open circles indicate unmethylated, and filled circles indicate methylated CpG dinucleotides. (<b>F</b>) Hematoxylin and eosin staining of teratoma sections of iPSC(SK46) clone 2. Teratomas were obtained from all iPSC clones. Left panel: neural tissue (ectoderm); middle panel: cartilage (mesoderm); right panel: gut epithelium (endoderm). Scale bars: 100 µm.</p>