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sc-12-0502_sm_SupplFigure1.pdf723KFigure S1. Detection of genomic rearrangement by Southern blot The locations of probes and restriction enzyme recognition sites used for the Southern blot analyses at the TRD locus (A) and IGH locus (B). C. The Southern blot analyses of the TRD locus. Genomic DNA (6 μg) was extracted from human ES cells (KhES3) and iPS cells, digested with Hind III and analyzed for V(D)J rearrangements at the TRD (T-cell receptor delta) locus by a Southern blotting analysis using a TRD probe. Cells were grown in non-T (NTm) and T cell (Tm) media, as indicated. The open arrowheads indicate bands derived from the germline allele. The absence of the band in clones 585A1, 585B1, 604A1, and 604B1 indicates the excision of the TRD region due to the rearrangement of the TRA loci. No indication of TRD recombination was observed in clones (692D2, 692E1, 648A1, and 648B1) which were established using non-T cell medium. D. The results of the Southern blot analyses of the IGH locus. No indication of IGH recombination was observed for PBMC-derived iPS lines.
sc-12-0502_sm_SupplFigure2.tif966KFigure S2. Screening of enhancing factors in human fibroblasts A. iPSC induction was performed with human dermal fibroblasts by using 2.25 μg of Y3 combination and 0.75 μg of an additional plasmid, which encodes the indicated gene. DsRedExpress (DsRed) was used as a negative control. The number of iPSC colonies was counted 25 days after the transfection. B. iPSC induction was carried out with 1.8 μg of the Y3 or Y4 combination with 0.6 μg GLIS1 and/or a TP53 shRNA expression vector. To adjust the total amount of plasmid to 3 μg, the DsRedExpress expression vector was added.
sc-12-0502_sm_SupplFigure3.tif967KFigure S3. Expression vectors in the Y4+EBNA combination.pCXWB-EBNA1 encodes EBNA1 under the control of CAG promoter, but lacks OriP sequence.CAG, CAG promoter; WPRE, woodchuck hepatitis post-transcriptional regulatory element; shRNA, shRNA against TP53; and pA, polyadenylation signal.
sc-12-0502_sm_SupplFigure4.tif2370KFigure S4. Expression of EGFP fluorescence from an episomal vector. Human fibroblasts were transduced with an episomal vector encoding EGFP with or without an extra EBNA1 expression vector via electroporation. The cells were analyzed by flow cytometry 6, 14, 22, and 30 days after the transduction.
sc-12-0502_sm_SupplFigure5.tif401KFigure S5. Effects of the extra EBNA1 vector on iPSC induction from human fibroblasts.Combinations of plasmids were introduced into human adult fibroblasts (HDF1388). MEFs and SNL cells were used as feeder cells.
sc-12-0502_sm_SupplFigure6.tif1426KFigure S6. Genomic PCR of iPSC clones. Genomic DNA was isolated from PMNC- and fibroblast-derived iPSC clones, and was used for PCR analysis. PMNC-iPSC clones were established with either Y4 or Y4 + EBNA1 (Y4E) from donor #1 in non-T (NTm) or T cell (Tm) medium. PCR primer sets were designed to detect transgenes of indicated regions. In the lanes labeled FBXO15, PCR primers only detected endogenous gene as a loading control. The plasmids equivalent to 4, 1, 0.25, and 0 copies were added to human genome for control detection (x4, x1, x0.25, and x0, respectively).
sc-12-0502_sm_SupplFigure7.pdf373KFigure S7. Effects of the extra EBNA1 vector on iPSC induction from PMNCs PMNCs were transduced by the Y4 combination with or without the additional EBNA1 expression vector. After the transduction, 1.0 and 0.3 million of the cells were seeded in non-T (NTm) and T cell (Tm) medium, respectively. The ESC-like cells were detected by alkaline phosphatase staining on day 22.
sc-12-0502_sm_SupplFigure8.tif2141KFigure S8. Clonality of iPSCs based on detection of genomic rearrangement in the TRB locus A. The results of the Southern blot analyses of the TRB locus. Genomic DNA (6 μg) was extracted from human dermal fibroblasts (HDF) and iPS cells, digested with Hind III and analyzed for V(D)J rearrangements at the TRB locus by a Southern blotting analysis. The open arrowheads indicate bands derived from the germline allele. B. Representative data regarding the characterization of the TRB locus by a PCR-based method. The genomic rearrangements between Vβ and Jβ2 were detected using multiplex primers. Blue lines show the signal from the amplified fragments indicating that clones #3, 6, and 10 had the same origin, whereas #9 was derived from different T cells. The red lines indicate the size marker. The PCR analysis also revealed that iPSC clones #5 and 8, but not #15, were derived from a single origin, and that clones #12 and 9 had different rearrangement patterns.
sc-12-0502_sm_SupplFigure9.tif1834KFigure S9. Characterization of iPSC clones. The expression of pluripotent cell marker genes detected by an RT-PCR analysis. Total RNA was isolated from CB- and PMNC-iPSC clones. PMNC-iPSC clones were established with either Y4 or Y4 + EBNA1 (Y4E) from two individual cells in non-T (NTm) or T cell (Tm) medium. Retrovirus-derived iPSC clones (201B7) and ESC lines (KhES-3 and H9) were also examined. In the lanes labeled OCT3/4 and SOX2, PCR primers only detected endogenous gene expression. G3PDH was used as a loading control. Total RNA was isolated from PMNCs as a negative control.
sc-12-0502_sm_SupplFigure10.pdf1464KFigure S10. Teratomas derived from iPSCs and eosin staining of teratomas derived from the iPSC clones established with Y4 and the additional EBNA1 vector. Bar = 100 μm.
sc-12-0502_sm_SupplTable1.tif181KSupplementary Table 1
sc-12-0502_sm_SupplTable2.tif171KSupplementary Table 2
sc-12-0502_sm_SupplTable3.tif60KSupplementary Table 3
sc-12-0502_sm_SupplTable4.tif466KSupplementary Table 4
sc-12-0502_sm_SupplTable5.tif118KSupplementary Table 5
sc-12-0502_sm_SupplTable6.tif121KSupplementary Table 6
sc-12-0502_sm_SupplTable7.tif151KSupplementary Table 7
sc-12-0502_sm_SupplTable8.tif40KSupplementary Table 8

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