Telephone: +81-3-5363-3475; Fax: +81-3-5363-3475
Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 25 FEB 2013
Copyright © 2012 AlphaMed Press
Volume 31, Issue 3, pages 479–487, March 2013
How to Cite
Nagamatsu, G., Kosaka, T., Saito, S., Honda, H., Takubo, K., Kinoshita, T., Akiyama, H., Sudo, T., Horimoto, K., Oya, M. and Suda, T. (2013), Induction of Pluripotent Stem Cells from Primordial Germ Cells by Single Reprogramming Factors. STEM CELLS, 31: 479–487. doi: 10.1002/stem.1303
Author contributions: G.N.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing.; T. Kosaka: conception and design, collection and assembly of data, and data analysis and interpretation; S.S.: performed informatics analysis of microarray data; H.H.: generated and analyzed the chimeric mice of mPGCs; K.T.: conception and design and data analysis and interpretation.; T. Kinoshita: collection and assembly of data and data analysis and interpretation; H.A. and T. Sudo: performed the microarray experiments; K.H.: performed informatics analysis of the microarray data; M.O.: administrative support and data analysis and interpretation; T. Suda: conception and design, financial support, administrative support, data analysis and interpretation, and final approval of the manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS December 19, 2012.
- Issue online: 25 FEB 2013
- Version of Record online: 25 FEB 2013
- Accepted manuscript online: 19 DEC 2012 02:04AM EST
- Manuscript Accepted: 27 NOV 2012
- Manuscript Received: 21 MAR 2012
- PRESTO of the Japan Science and Technology Agency and Scientific Research
- MEXT to Keio University and the Keio University Medical Science Fund
Additional Supporting Information may be found in the online version of this article.
|sc-12-0279_sm_SupplFigure1.tif||614K||Supplementary Figure S1. The culture method used to induce iPS cells from PGCs. The gonads of E11.5 Nanog-GFP transgenic embryos were collected and single cell suspensions were generated by trypsin treatment. PGCs were isolated by FACS sorting based on GFP fluorescence. The collected PGCs were seeded on SCF-expressing m220 feeder cells and infected with viruses containing various combinations of iPS cell induction factors.|
|sc-12-0279_sm_SupplFigure2.tif||276K||Supplementary Figure S2. Induction of mPGCs by factors used to induce iPS cells. The number of ES cell-like colonies observed 10 days after 2,500 PGCs were induced with the indicated factor(s). The results from two independent experiments are shown. Factors used to induce mPGCs (shown along the horizontal axis) are designated as follows: O, Oct3/4; K, Klf4; S, Sox2; M, c-Myc.|
|sc-12-0279_sm_SupplFigure3.pdf||47K||Supplementary Figure S3. EB formation from mPGCs. EBs generated from mPGCs induced with the indicated factors under non-adherent culture conditions.|
|sc-12-0279_sm_SupplFigure4.pdf||89K||Supplementary Figure S4. Morphology of mPGCs cultured in 2i +LIF or bFGF + Activin. (Upper) mPGCs induced with the indicated factors and cultured in the presence of 2i (MEK inhibitor and GSK-3β inhibitor) + LIF or (lower) bFGF + Activin, observed by phase contrast microscopy. Scale bars, 100μM.|
|sc-12-0279_sm_SupplFigure5.tif||1157K||Supplementary Figure S5. Generation of chimeric mice from mPGCs. mPGCs were injected into blastocysts of ICR mice. The contribution of chimeric mice was estimated from the coat color of the new-born mice. Arrowhead indicated a colored coat, which were due to mPGCs.|
|sc-12-0279_sm_SupplFigure6.tif||396K||Supplementary Figure S6. Nanog-GFP expression in mPGCs. GFP expression driven by the Nanog promoter in mPGCs induced with various factors was compared with that in E11.5 PGCs.|
|sc-12-0279_sm_SupplFigure7.tif||2080K||Supplementary Figure S7. Southern hybridization of mPGCs. Southern hybridization to monitor virus integration in mPGCs infected with viruses containing various induction factors. Genomic DNA was digested with EcoRV (a) or XhoI (b) and detected with a virus vector-specific probe. Factors used to induce mPGCs are designated as follows: O, Oct3/4; K, Klf4; S, Sox2; M, c-Myc. 4F iPS cells (TTF) induced from mouse tail tip fibroblasts by all four factors were used as a positive control.|
|sc-12-0279_sm_SupplFigure8.tif||322K||Supplementary Figure S8 Expression of virally-introduced factors in pluripotent candidate cells. PGCs were infected with viruses containing the indicated factors. Two days after infection, the pluripotent candidate cells were purified based on expression of Nanog-GFP and SSEA-1. The expression of virally-introduced factors were analyzed using specific sets of primers and probes (mean ± SD; three independent experiments, n = 6). **P<0.01.|
|sc-12-0279_sm_SupplFigure9.tif||297K||Supplementary Figure S9 Fluorescence of pluripotent candidate cells following infection with a virus expressing DsRed. PGCs were infected with a virus containing DsRed-Express. Two days after infection, the pluripotent candidate cells were purified based on expression of Nanog-GFP and SSEA-1. DsRed fluorescence was analyzed by flow cytometry.|
|sc-12-0279_sm_SupplFigure10.pdf||78K||Supplementary Figure S10. Induction of mPGCs with L-Myc. a. The number of ES cell-like colonies observed 10 days after infection of 2,500 PGCs with a virus containing L-Myc. The results from two independent experiments are shown. b, c. Typical morphology of established mPGCs observed by phase contrast microscopy (b) and Nanog-GFP fluorescence (c). Scale bars, 100μM. d, e. Gene expression profiles of germ cell markers in mPGCs compared with those of parental E11.5 PGCs. Relative gene expression in mPGCs is shown for Mvh (d) and Blimp-1 (e). f-k. Gene expression profiles of pluripotent cell markers in mPGCs compared with those of ES cells (TT2). Relative gene expression in mPGCs is shown for ECAT1 (f), Eras (g), Klf4 (h), Sox2 (i), Oct3/4 (j), and Nanog (k).|
|sc-12-0279_sm_SupplFigure11.pdf||68K||Supplementary Figure S11. Differentiation capacity and Nanog-GFP expression of mPGCs induced with L-Myc. a-c. Teratoma formation by mPGCs induced with L- Myc. Ectoderm (a), mesoderm (b), and endoderm (c) are shown. Scale bars, 200μM. d. EBs formed from mPGCs induced by L-Myc under non-adherent culture conditions. e. GFP expression driven by the Nanog promoter in mPGCs generated by L-Myc was compared with that in E11.5 PGCs.|
|sc-12-0279_sm_SupplFigure12.tif||469K||Supplementary Figure S12. Total expression levels of the reprogramming factors in pluripotent candidate cells at 2 days after infection. a-c. Total expression levels (endogenous and virus-derived) of Sox2, Oct3/4, and c-Myc in PGCs at 2 days after infection with viruses containing various factors compared with their expression levels in parental E11.5 PGCs and bFGF-treated EG cells. Cells were sorted by FACS based on the expression of Nanog-GFP and SSEA-1. Relative gene expression is shown (mean ± SD; three independent experiments, n = 6). **P<0.01, *P<0.05.|
|sc-12-0279_sm_SupplTable1.pdf||27K||Supplementary Table 1|
|sc-12-0279_sm_SupplTable2.xls||585K||Supplementary Table 2|
|sc-12-0279_sm_SupplTable3.xls||426K||Supplementary Table 3|
|sc-12-0279_sm_SupplTable4.xls||7452K||Supplementary Table 4|
|sc-12-0279_sm_SupplTable5.pdf||34K||Supplementary Table 5|
|sc-12-0279_sm_SupplTable6.pdf||28K||Supplementary Table 6|
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.