Undifferentiated State of Induced Pluripotent Stem Cells after Long-Term Culture using Automated Cell Culture Device. (A) Alkaline phosphatase activity of iPS cells at 0 and 17 passages. Scale: 100 μm. (B) Immunostaining of the pluripotency markers SSEA-1 and SSEA-3 in iPS cells at 0 and 17 passages. Scale: 100 μm. (C) Quantitative PCR analysis of the gene expression of the stem cell markers Oct3/4, Sox2, and Nanog, and the fibroblast marker CD13 compared with the MEFs. (D) Quantitative PCR analysis of the gene expression in EBs derived from automatically cultured iPS cells. The expressions of pluripotency markers; Zfp42, Nanog, endoderm markers; Ttr, Afp, mesoderm markers; Myh6, Brachyury, and ectoderm markers; Gfap, Nes, were compared with the iPS cells. *Statistically significant (p <0.05).