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

(A) Bright-field image of iPSCs derived from human adult skin fibroblasts (iPS(SK46) clone 2). Scale bar: 100 µm. (B) G-banding chromosome analysis of iPS(SK46) clone 2 (p17). (C) 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. T-OCT4: transgene OCT4; T-SOX2: transgene SOX2; T-NANOG: transgene NANOG; T-LIN28: transgene LIN28; T-c-MYC: transgene c-MYC; T1-KLF4: transgene KLF4 (1); T2-KLF4: transgene KLF4 (2); T-SV40LT: transgene SV40LT; OCT4: endogenous OCT4. 32 PCR cycles were used for all primer sets. (D) Quantitative RT-PCR analysis of the endogenous OCT4, NANOG, SOX2 and LIN28 expression in iPSC clones. Data shown are mean ± SEM (n = 3). (E) Bisulfite-sequencing analysis of the methylation status of the OCT4 and NANOG promoters in iPSC clones. Open circles indicate unmethylated, and filled circles indicate methylated CpG dinucleotides. (F) 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.