Analysis of pluripotency of iPS cells generated by plasmid transfection. A) Micrographs comparing the morphology of the plasmid-derived iPS cells (iPSK3) and human ES cell line H9.14 cultured on mitotically inactivated MEFs and alkaline phosphatase activity identified by histochemistry (Scale bar = 100 μm). B) Immunostaining revealing the presence of OCT4 (red) and SSEA4 (green) in plasmid-derived iPSK3 cells and control H9 human ES cells. Cell nuclei were detected using DAPI stain (Blue) (Scale bar = 100 μm). C) Representative FACS analysis demonstrating that ≥ 99% of iPSK3 cells in culture (passage 8-10) express markers of pluripotency including OCT4, SSEA4, TRA1-60 and TRA1-81. Cells expressing the fibroblast marker CD13 were not detected. D) Micrographs of H & E stained sections through teratomas that formed from iPSK3 cells after injection into immune deficient mice. Cell types derived from all three germ layers - ectoderm, endoderm and mesoderm (indicated with *) - were detected (Scale bar = 100 μm). E) Karyotype of iPSK3 cells revealed a normal distribution of 46 chromosomes with XY sex chromosomes. Chromosomal rearrangements were not detected by G-banding. F) STR analyses using CODIS primers demonstrated that the DNA fingerprint of iPSK3 cells was indistinguishable from that of CRL2097 foreskin fibroblasts used as recipients for reprogramming.