Additional Supporting Information may be found in the online version of this article.

STEM_685_sm_SuppInfo.doc29KSupporting Information
STEM_685_sm_SuppFigS1.eps3719KSupporting Information Figure S1. Teratoma formation
STEM_685_sm_SuppFigS2.eps2850KSupporting Information Figure S2. Germline transmission/competency of 2A iPSCs. (A) F1 mice derived from 2A-4F-1 iPSCs. (B) F1 mice derived from 2A-4F-33 iPSCs. (C) A 90% chimeric mouse (9 days old) generated using the 2A-3F-EGFPtg-4 clone. 2A-3F-EGFPtg-4 iPSCs harbor the EGFP gene under the control of a ubiquitous promoter. (D) Testes from the chimeric mouse shown in (C). (E) Seminiferous tubule from the testes shown in (D).
STEM_685_sm_SuppFigS3.eps2796KSupporting Information Figure S3. Examples of various levels of chimerism as defined by coat color
STEM_685_sm_SuppFigS4.eps1588KSupporting Information Figure S4. Chimeric mouse generation test for iPSCs established using another procedure and another mouse strain. (A) iPSCs generated using a retroviral gene transduction system. (B) iPSCs generated from MEFs derived from Nanog-GFP tg mice. p, germline transmission positive; n, germline transmission negative.
STEM_685_sm_SuppFigS5.eps3498KSupporting Information Figure S5. Abnormal embryos developed from 3F-iPSCs
STEM_685_sm_SuppFigS6A.eps9380KSupporting Information Figure S6. Blastocysts at 48 hours post-aggregation. (A) 2A-4F-EGFPtg-1.
STEM_685_sm_SuppFigS6B.eps8984KSupporting Information Figure 6B. (B) 2A-3F-EGFPtg-4.
STEM_685_sm_SuppFigS6C.eps9545KSupporting Information Figure 6C. (C) 2A-3F-EGFPtg-15. Merged, bright field and fluorescence images are shown.
STEM_685_sm_SuppFIgS6D.eps1003KSupporting Information Figure 6D. (D)Reduction rate of GFP signals after aggregation. The results of in vitro blastocyst formation assays of 2A-4F-EGFPtg-1, and 2A-3F-EGFPtg-4, -5, -7, -14 and -20 are shown. Twenty embryos were examined for each clone. The differences in the GFP signals between the 24 h later and at 72 h later timepoints are indicated. Standard errors are shown.
STEM_685_sm_SuppFigS7Aai.eps24442KSupporting Information Figure S7. (A) Bisulfite sequencing analysis of pluripotent marker genes. Data were analyzed using QUMA software. Open circles indicate unmethylated CpGs, whereas closed circles indicate methylated CpGs.
STEM_685_sm_SuppFigS7B.eps1450KSupporting Information Figure 7B. (B) Real-time PCR analysis of imprinting genes in iPSCs. The data were normalized using Gapdh gene expression. The relative expression levels compared with each transcript in the R1 cells are presented. Standard deviations (SD) are shown (n=3).
STEM_685_sm_SuppFigS8.eps852KSupporting Information Figure S8. Chromosome analysis of iPSCs. The frequency of cells with 40 chromosomes is shown. Standard errors (SE) are also shown.
STEM_685_sm_SuppFigS9.docx164KSupporting Information Figure S9. Differentially expressed genes between 2A-4F-iPSs and 2A-3F-iPS-4 screened via the HiCEP transcriptome analysis method. A total of 20,138 transcripts including unknown mRNAs were analyzed and capillary electropherograms are shown. Red and blue lines indicate the expression patterns for 2A-4F-1 and 2A-4F- 33, and for 2A-3F-1, 2A-3F-3 and 2A-3F-4, respectively. The Y-axis indicates the transcript levels and the X-axis indicates the length of the fragments [8]. Duplicate experiments with each cell line were performed. Information for each peak is provided in the table.
STEM_685_sm_SuppMovieS1.avi19940KSupporting Information Movie S1. Aggregation of 2A-4F-EGFPtg-1 cells
STEM_685_sm_SuppMovieS2.avi19929KSupporting Information Movie S2. Aggregation of 2A-3F-EGFPtg-4 cells
STEM_685_sm_SuppMovieS3.avi19934KSupporting Information Movie S3. Aggregation of 2A-3F-EGFPtg-15 cells

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