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. 2008 Nov 7;322(5903):945-9.
doi: 10.1126/science.1162494. Epub 2008 Sep 25.

Induced pluripotent stem cells generated without viral integration

Matthias Stadtfeld  1 , Masaki Nagaya, Jochen Utikal, Gordon Weir, Konrad Hochedlinger
Affiliations

Induced pluripotent stem cells generated without viral integration

Matthias Stadtfeld et al. Science. .

Abstract

Pluripotent stem cells have been generated from mouse and human somatic cells by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc. A major limitation of this technology is the use of potentially harmful genome-integrating viruses. We generated mouse induced pluripotent stem (iPS) cells from fibroblasts and liver cells by using nonintegrating adenoviruses transiently expressing Oct4, Sox2, Klf4, and c-Myc. These adenoviral iPS (adeno-iPS) cells show DNA demethylation characteristic of reprogrammed cells, express endogenous pluripotency genes, form teratomas, and contribute to multiple tissues, including the germ line, in chimeric mice. Our results provide strong evidence that insertional mutagenesis is not required for in vitro reprogramming. Adenoviral reprogramming may provide an improved method for generating and studying patient-specific stem cells and for comparing embryonic stem cells and iPS cells.

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Figures

Figure 1
Figure 1. Analysis of pluripotency markers in Adeno-iPS cells
(A) Brightfield (upper panel) and fluorescence (lower panel) images of an Adeno-iPS cell clone established from Sox2-GFP fetal liver cells taken at passage 0 (P0) and passage 2 (P2). (B) Expression of endogenous c-myc, Klf4, Oct4, Sox2 and nanog measured by qPCR in Adeno-iPS cells derived from fetal liver (FL), fibroblasts (TTF) and hepatocytes (HEP) as well as in V6.5 control ES cells. (C) Bisulfite sequencing of the Oct4 and Nanog promoters in hepatocytes, ES cells and iPS cells derived from hepatocytes. Open circles represent unmethylated CpGs; closed circles denote methylated CpGs. (D) Expression levels of endogenous gapdh (G) as well as adenoviral c-myc (M), Klf4 (K), Oct4 (O) and Sox2 (S) in fibroblasts three days after infection with adenoviruses (TTF + 4 adenos), ES cells and Adeno-iPS cells derived from fetal liver, fibroblasts and hepatocytes. Error bars indicate one standard deviation. The absence of a bar indicates that the respective cDNA was not detected.
Figure 2
Figure 2. Absence of viral integration in Adeno-iPS cell
(A) Schematic drawing of the adenoviral vector indicating the position of the cDNA and the sizes of the respective DNA fragments after BamHI digestion. The bracketed BamHI site is only present in the Oct4 cDNA. A pBluescript (pBS) sequence present in both the adenoviral vector and the Oct4IND transgene is highlighted. (B) PCR analyses for adenoviral integration in genomic DNA from the indicated Adeno-iPS clones as well as from V6.5 ES cells that served as a negative control (−). Arrowhead indicates the position of the positive control band amplified from vector DNA (+). (C) Southern blot analysis of BamHI-digested genomic DNA using DNA fragments encompassing the entire adenoviral vector backbone isolated from pAd-Oct4 as probes. Plasmid DNA of pAd-Klf4 and pAd-Oct4 diluted to the equivalent of 0.2, 1 or 2.5 integrations per genome and genomic DNA of HEK cells (which contain adenoviral sequences in their genome) were used as positive controls. An asterisk (*) indicates bands resulting from hybridization of the pBS sequence in the adenoviral probe to transgenic sequences in the Oct4IND allele. Note that these bands are absent in HEP iPS, V6.5 ES and HEK cells. A double asterisk (**) indicates bands resulting from hybridization of parts of the probe to sequences present in the endogenous Oct4 locus or in Oct4 pseudogenes. These bands are present in all lanes containing genomic DNA including the wild type control and therefore serve as loading controls. Solid arrowheads indicate the position of BamHI fragments of the adenoviral vector and open arrowheads highlight adenoviral sequences detected in HEK cells.
Figure 3
Figure 3. Pluripotency of Adeno-iPS cell
(A–C) Images of histological sections through teratomas formed by Adeno-iPS cells subjected to hematoxylin and eosin staining, showing keratinized epithelium (A), mucous epithelium (B) and cartilage (C). (D–I) Fluorescence images showing the contribution of red fluorescent protein-labeled Adeno-iPS cells to lung, brain and heart in a postnatal chimeric animal. Nuclei were counterstained with DAPI (blue). The small insets in (D, F, H) highlight the fields magnified in (E, G, I) whereas the insets in (E, G, I) show the background fluorescent levels and DAPI staining of corresponding tissues in a non-chimeric littermate. (J, K) Images of coat-color chimeras derived from fetal liver (J) and hepatocytes (K) Adeno-iPS cells. (L–O) Fluorescence and brightfield images of a wild type (L, M) blastocyst and an Oct4-GFP (N, O) blastocyst obtained after mating a chimeric mouse made with TTF-1 iPS cells expressing GFP from the Oct4 promoter with a wild type female.
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