Telephone: 81-75-366-7042; Fax: 81-75-366-7098
Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 25 FEB 2013
Copyright © 2012 AlphaMed Press
Volume 31, Issue 3, pages 458–466, March 2013
How to Cite
Okita, K., Yamakawa, T., Matsumura, Y., Sato, Y., Amano, N., Watanabe, A., Goshima, N. and Yamanaka, S. (2013), An Efficient Nonviral Method to Generate Integration-Free Human-Induced Pluripotent Stem Cells from Cord Blood and Peripheral Blood Cells. STEM CELLS, 31: 458–466. doi: 10.1002/stem.1293
Author contributions: K.O.: conception and design, financial support, collection and/or assembly of data, provision of study material, data analysis and interpretation, manuscript writing, and final approval of manuscript; T.Y.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; Y.M. and Y.S.: collection and/or assembly of data and provision of study material; N.A. and A.W.: collection and/or assembly of data and data analysis and interpretation; N.G.: provision of study material; S.Y.: conception and design, financial support, and manuscript writing.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS November 29, 2012.
- Issue online: 25 FEB 2013
- Version of Record online: 25 FEB 2013
- Accepted manuscript online: 29 NOV 2012 05:50AM EST
- Manuscript Accepted: 7 NOV 2012
- Manuscript Received: 29 MAY 2012
- Program for Promotion of Fundamental Studies in Health Sciences of National Institute of Biomedical Innovation
- Leading Project of Ministry of Education, Culture, Sports, Science and Technology (MEXT)
- Program for World-Leading Innovative Research and Development on Science and Technology (FIRST Program) of Japan Society for the Promotion of Science
- Grants-in-Aid for Scientific Research of Japan Society for the Promotion of Science and MEXT
- Grants-in-Aid for Scientific Research for Young Scientists B
Additional Supporting Information may be found in the online version of this article.
|sc-12-0502_sm_SupplFigure1.pdf||723K||Figure 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.tif||966K||Figure 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.tif||967K||Figure 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.tif||2370K||Figure 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.tif||401K||Figure 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.tif||1426K||Figure 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.pdf||373K||Figure 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.tif||2141K||Figure 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.tif||1834K||Figure 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.pdf||1464K||Figure 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.tif||181K||Supplementary Table 1|
|sc-12-0502_sm_SupplTable2.tif||171K||Supplementary Table 2|
|sc-12-0502_sm_SupplTable3.tif||60K||Supplementary Table 3|
|sc-12-0502_sm_SupplTable4.tif||466K||Supplementary Table 4|
|sc-12-0502_sm_SupplTable5.tif||118K||Supplementary Table 5|
|sc-12-0502_sm_SupplTable6.tif||121K||Supplementary Table 6|
|sc-12-0502_sm_SupplTable7.tif||151K||Supplementary Table 7|
|sc-12-0502_sm_SupplTable8.tif||40K||Supplementary Table 8|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.