Nature Methods | Brief Communication

A more efficient method to generate integration-free human iPS cells

Journal name:
Nature Methods
Volume:
8,
Pages:
409–412
Year published:
DOI:
doi:10.1038/nmeth.1591
Received
Accepted
Published online

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ~20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

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  1. Establishment of human iPSCs.
    Figure 1: Establishment of human iPSCs.

    (a) Combinations of reprogramming factors and episomal vectors used in this study. (b) Episomal expression vectors in the Y4 combination. CAG, CAG promoter; WPRE, woodchuck hepatitis post-transcriptional regulatory element; and pA, polyadenylation signal. (c) Schematic of the pla-iPSC induction protocol. DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; MSCGM, mesenchymal stem cell growth medium; bFGF, basic fibroblast growth factor. (d) Numbers of colonies per 1.0 × 105 cells obtained with different combinations of reprogramming factors. Control, cells transduced with episomal vector encoding EGFP; MEF, mouse embryonic fibroblasts; SNL, mouse embryonic fibroblast cell line. Data are means ± s.d. of numbers of ESC-like colonies obtained from 15 independent induction experiments using five cell lines. ****P < 0.05 against T1, T2, T3 and control; ***P < 0.05 against T1, T3 and control; **P < 0.05 against T1 and control; *P < 0.05 against control.

  2. Characterization of pla-iPSC clones.
    Figure 2: Characterization of pla-iPSC clones.

    (a,b) Phase contrast images of an established pla-iPSC line. Scale bars, 1 mm (a) and 100 μm (b). (c) RT-PCR analyses for pluripotent cell markers. Total RNA was isolated from pla-iPSC clones established with the Y1 (clone 454B-1), Y2 (454C-2), Y3 (454D-1) or Y4 (454E-2, 451F-3, 457C-1 and 453F-2) combinations, from retrovirus-derived iPSC clones (retro-iPSC) and from ESC lines. In the lanes labeled OCT3/4 and SOX2, PCR primers only detected endogenous gene expression; in the Ret-OCT lane, PCR primers specifically amplified the retroviral OCT3/4 transgene. GAPDH was used as a loading control. As a negative control, GAPDH amplification was also performed without reverse transcription (no RT). Fibroblasts 4 d after electroporation of the Y4 mixture (HDF-elepo) and mouse embryonic fibroblast cell line (SNL) were used as other negative controls. (d) DNA methylation status of the NANOG promoter region in the indicated cell lines. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. (e) Copy numbers of episomal vectors in pla-iPSC clones. Numbers in parentheses indicate passage number. Also shown are the estimated numbers of cells analyzed for each clone. Fibroblasts 6 d after electroporation of the Y4 combination were analyzed (fibro-d6) as a positive control. (f–h) Differentiation of pla-iPSC clone (454E-2) into dopaminergic neurons. Micrographs are immunostained for Tuj1 (f) and tyrosine hydroxylase (TH) (g). A merged image with nuclear staining using DAPI (h) is shown. Scale bars, 20 μm. (i,j) Differentiation of pla-iPS clone (454E-2) into retinal pigment epithelial cells. Scale bars, 100 μm (i) and 50 μm (j).

  3. Estimated coverage of the Japanese population by HLA homozygous donors.
    Figure 3: Estimated coverage of the Japanese population by HLA homozygous donors.

    (a) Estimated cumulative coverage of the Japanese population by theoretical unique HLA homozygous donors at HLA-A, HLA-B and HLA-DRB1 loci with four-digit specification. (b) Estimated numbers of donors required to identify individuals with unique HLA homozygous haplotypes.

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Author information

Affiliations

  1. Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.

    • Keisuke Okita,
    • Yasuko Matsumura,
    • Yoshiko Sato,
    • Aki Okada,
    • Asuka Morizane,
    • Hyenjong Hong,
    • Masato Nakagawa,
    • Koji Tanabe,
    • Masayo Takahashi,
    • Jun Takahashi &
    • Shinya Yamanaka

  2. Department of Biological Repair, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

    • Asuka Morizane &
    • Jun Takahashi

  3. Laboratory for Retinal Regeneration, Center for Developmental Biology, RIKEN, Kobe, Japan.

    • Satoshi Okamoto &
    • Masayo Takahashi

  4. Department of Tissue and Organ Development, Gifu University Graduate School of Medicine, Gifu, Japan.

    • Ken-ichi Tezuka &
    • Takahiro Kunisada

  5. Department of Oral and Maxillofacial Science, Gifu University Graduate School of Medicine, Gifu, Japan.

    • Toshiyuki Shibata

  6. Human Leukocyte Antigen (HLA) Laboratory, Kyoto, Japan.

    • Hiroh Saji

  7. Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan.

    • Shinya Yamanaka

  8. Yamanaka Induced Pluripotent Stem Cell Project, Japan Science and Technology Agency, Kawaguchi, Japan.

    • Shinya Yamanaka

  9. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA.

    • Shinya Yamanaka

Contributions

K.O. and S.Y. conceived the project and wrote the manuscript. K.O. constructed the vectors with H.H., M.N. and K. Tanabe, and conducted most of the experiments with Y.M., Y. S. and A.O. A.M. and J.T. carried out the differentiation experiment into dopaminergic neurons. S.O. and M.T. performed differentiation into retinal pigment epithelial cells. K. Tezuka., T.S. and T.K. established dental pulp cell lines. H.S. performed HLA haplotyping in Japanese population and supervised HLA analysis.

Competing financial interests

K.O., M.N. and S.Y. are filing a patent application to Japan, US and EU based on the results reported in this paper (PCT/JP2010/063733). S.Y. is a member of Scientific Advisory Board for iPS Academia Japan Inc. and iPierian Inc., which manage the patents.

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Supplementary information

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  1. Supplementary Text and Figures (2M)

    Supplementary Figures 1–9 and Supplementary Tables 1–7 and 9, 10

Excel files

  1. Supplementary Table 8 (406K)

    Haplotype frequency for HLA-A, HLA-B and HLA-DRB1 loci in Japanese population.

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