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Induced Pluripotent Stem Cells Generated Without Viral Integration

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Science  07 Nov 2008:
Vol. 322, Issue 5903, pp. 945-949
DOI: 10.1126/science.1162494

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.

The introduction of defined transcription factors into mouse and human somatic cells has recently been shown to reprogram the developmental state of mature cells into that of pluripotent embryonic cells, generating so-called “induced pluripotent stem (iPS) cells” (1). iPS cells have been generated from multiple cell types by viral expression of Oct4 and Sox2, combined with either Klf4 and c-Myc (111) or LIN28 and Nanog (12). iPS cells are molecularly and functionally highly similar to ES cells, which makes in vitro reprogramming an attractive approach to produce patient-specific stem cells for studying and potentially treating degenerative disease. Indeed, reprogrammed skin cells have recently been shown to alleviate the symptoms of Parkinson's disease (13) and sickle cell anemia (14) in mouse models. However, a major limitation of this technology is the use of viruses that integrate into the genome and are associated with the risk of tumor formation due to the spontaneous reactivation of the viral transgenes (8). The low efficiency of reprogramming (0.01 to 0.1% of input cells) also raised the possibility that insertional mutagenesis may be a prerequisite for in vitro reprogramming (15). For example, the retroviral tagging of explanted hematopoietic stem cells has been previously shown to select for clones in which the retroviral construct had inserted proximal to self-renewal genes and thus causes their activation (16). Whereas the sequencing of a limited number of insertion sites in iPS cells did not reveal common targets (17), this possibility has not been unequivocally ruled out yet (15).

Therefore, we set out to generate iPS cells from mouse somatic cells using adenoviral vectors that allow for transient, high-level expression of exogenous genes without integrating into the host genome (18). Specifically, we cloned the cDNAs for Oct4, Sox2, c-Myc, and Klf4 into replication-incompetent adenoviral vectors under the control of the human cytomegalovirus immediate early (hCMV IE) promoter (fig. S1). Initial attempts to reprogram mouse tail-tip fibroblasts (TTFs) into iPS cells with adenoviruses expressing the four reprogramming factors were unsuccessful, possibly owing to the rapid dilution of the virally encoded proteins from the cells. We have previously shown that viral Oct4 expression can be replaced by a doxycycline-inducible Oct4 allele driven by a reverse-tetracycline–dependent transactivator (rtTA) present in the ROSA26 locus (Oct4IND) (6, 19), and it has been reported that liver cells require lower numbers of viral integrations than fibroblasts to be reprogrammed (17). Therefore, we infected ∼500,000 adherent Oct4IND mouse fetal liver cells with adenoviruses expressing Sox2, Klf4, and c-Myc at multiplicities of infection (MOI, number of viral particles per cell) of 20 to 50. This led to an infection efficiency of about 40 to 50% of cells with each factor and an estimated infection efficiency of 20 to 30% of cells expressing all three adenoviral transgenes (fig. S2, A and E). After culture of infected fetal liver cells for 24 to 30 days in the presence of doxycycline, nine iPS-like colonies emerged that expressed the pluripotency markers Sox2 and SSEA-1 and could be expanded into stable ES cell-like lines, similar to iPS cells produced with retro- or lentiviral vectors (5, 20) (Fig. 1A). These adenoviral iPS (adeno-iPS) cells continued to grow in the absence of doxycycline, which indicated that transgenic Oct4 expression was no longer required.

Fig. 1.

Analysis of pluripotency markers in adeno-iPS cells. (A) Bright-field (top) and fluorescence (bottom) 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; filled 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 3 days after infection with adenoviruses (TTF + 4 adenos), ES cells and adeno-iPS cells derived from fetal liver, fibroblasts, and hepatocytes. Error bars indicate 1 SD. The absence of blue bars in (D) indicates that the respective cDNA was not detected.

These results prompted us to test if postnatal tail fibroblasts carrying the Oct4-inducible allele were equally amenable to reprogramming into adeno-iPS cells. Fibroblasts required MOIs of 50 to 250 to achieve an infection efficiency of ∼30% for each vector, resulting in an estimated 10 to 20% infection efficiency for triple-infected cells (fig. S2, B and E). Infection of more than 1,000,000 neonatal Oct4IND TTFs harboring an Oct4-GFP reporter with adenoviruses expressing myc, Klf4, and Sox2 in the presence of doxycycline gave rise to a single GFP+ colony that grew into a stable, doxycycline-independent line.

To identify an adult cell type that might not require transgenic Oct4 expression, we chose hepatocytes, which are highly permissive for adenoviral infection (20, 21). Indeed, MOIs of 1 to 4 were sufficient to infect 70 to 80% of these cells with individual vectors, with an estimated 50 to 60% of cells expressing all four viral reprogramming factors (fig. S2, C, D, and E). After incubation of ∼500,000 adult hepatocytes isolated from mice carrying the Oct4-GFP and ROSA26-rtTA alleles but lacking the Oct4-inducible allele with adenoviruses expressing c-myc, Klf4, Oct4, and Sox2, we obtained three colonies, all of which could be expanded into stable ES-like cell lines expressing the pluripotency markers Oct4 and SSEA-1 (fig. S3). Polymerase chain reaction (PCR) fingerprinting of adeno-iPS cells confirmed their derivation from hepatocytes rather than from a contaminating ES cell line (fig. S4). This demonstrates that iPS-like cells can be produced from adult cells by adenoviral vectors alone in the absence of the transgenic Oct4 allele.

Next, we tested whether adeno-iPS cells had reestablished pluripotency at the molecular level by examining the activity of ES cell–specific markers. Expression analysis for the endogenous Oct4, Klf4, Sox2, c-Myc, and Nanog genes by quantitative PCR (qPCR) gave signals that were indistinguishable from those of ES cells, consistent with faithful molecular reprogramming (Fig. 1B). In agreement with this, the Oct4 and Nanog promoters became demethylated in adeno-iPS cells to an extent similar to that seen in ES cells, whereas they remained hypermethylated in cultured fibroblasts and hepatocytes, which indicated that adeno-iPS cells had undergone successful epigenetic reprogramming (Fig. 1C and fig. S5). In contrast to freshly infected fibroblasts, viral transcripts could not be detected in any of the adeno-iPS cell lines tested, which suggested that the viral vectors had been diluted from the cells over time (Fig. 1D).

Adenoviral vectors can integrate into the genome of host cells at extremely low frequencies (22). To exclude the possibility of permanent viral integration, PCR analysis of genomic DNA isolated from adeno-iPS cell clones was performed with primers recognizing the different cDNA expression cassettes (see fig. S1 for the position of the sequences recognized by the primers). Whereas adenoviral vector DNA, used as a positive control, readily produced PCR signals, we were unable to amplify these PCR products from genomic DNA from any of the adeno-iPS cells (Fig. 2B). Southern blot analysis using the cDNAs of the four viral vectors as probes confirmed the PCR results and yielded no evidence for the continuous presence of the adenoviral sequences in the adeno-iPS cells whereas the single-copy Oct4 transgenic allele integrated into the Col1A locus could be readily detected in iPS cell clones generated from Oct4IND cells (fig. S6). To rule out genomic integration of adenoviral sequences other than the cDNAs, we performed Southern blot analysis using the Bam HI–digested full-length vector as a probe. Again, we did not detect exogenous viral sequences in the genomes of adeno-iPS cells, whereas adenoviral sequences were detectable in human embryonic kidney (HEK) cells, consistent with a previous report (23) (Fig. 2C). Moreover, the pBluescript (pBS)-derived portion of the adenoviral vector probe cross-hybridized with the Oct4 transgene, which also carries the pBS backbone, giving rise to a specific ∼3-kb signal in the iPS cell lines derived from fetal liver and TTFs and thus serving as an internal positive control (Fig. 2C). Together, these results strongly suggest that viral integration is not required for the generation of iPS cells. Although highly unlikely, we cannot rule out the possibility that small pieces of adenoviral DNA have inserted into the genome of adeno-iPS cells but were not observed because of the detection limits of Southern blot analysis.

Fig. 2.

Absence of viral integration in adeno-iPS cells. (A) Schematic drawing of the adenoviral vector indicating the position of the cDNA and the sizes of the respective DNA fragments after Bam HI digestion. The bracketed Bam HI 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 Bam HI–digested genomic DNA by using DNA fragments encompassing the entire adenoviral vector backbone isolated from pAd-Oct4 as probes. Plasmid DNA of pAd-Klf4 and pAd-Oct4 was 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. Filled arrowheads indicate the position of Bam HI fragments of the adenoviral vector, and open arrowheads highlight adenoviral sequences detected in HEK cells.

To ascertain the developmental potential of adeno-iPS cells, we injected the cells into the flanks of SCID mice. All cell lines tested produced teratomas after 3 to 4 weeks, which, upon histological examination, showed differentiation into representative cell types of the three germ layers including muscle, cartilage, and epithelial cells, thus demonstrating the pluripotency of adeno-iPS cells (Fig. 3, A to C; also see Table 1 and table S1 for a summary of all adeno-iPS lines). In addition, adeno-iPS cells generated apparently normal postnatal chimeras following injection into blastocysts. One chimera, obtained after blastocyst injection of adeno-iPS cells labeled with a lentivirus expressing the red fluorescent protein tdTomato, was killed at birth to examine the contribution of adeno-iPS cells to different tissues. As shown in Fig. 3, D to I, a high degree of chimerism was seen in multiple tissues including the lungs, brain, and heart. Adeno-iPS cells gave rise to high-degree coat color chimeras (Fig. 3, J and K) and differentiated into functional germ cells, as evidenced by the derivation of GFP+ blastocysts after breeding of a male chimera generated with TTF-derived adeno-iPS cells with a wild-type female (Fig. 3, L to O). Moreover, adult male chimeras derived from FL-9 and TTF-1 adeno-iPS cells gave rise to viable germline offspring that showed the iPS cell-specific agouti coat color after mating with BDF1 female mice (1/20 or 5% of FL-9 offspring and 11/11 or 100% of TTF-1 offspring). Together, these results indicate that adeno-iPS cells share the same developmental potential as iPS cells obtained with integrating viruses. Importantly, we have not observed tumor formation in any of the 12 coat color chimeras ranging up to 20 weeks of age.

Fig. 3.

Pluripotency of adeno-iPS cells. (A to 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 to 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 counter-stained with DAPI (blue). The small insets in (D), (F), and (H) highlight the fields magnified in (E), (G), and (I); the insets in (E), (G), (I) show the background fluorescent levels and DAPI staining of corresponding tissues in a nonchimeric littermate. (J and K) Images of coat-color chimeras derived from fetal liver (J) and hepatocytes (K) adeno-iPS cells. (L to O) Fluorescence and bright-field images of a wild-type (L and 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.

Table 1.

Derivation and characterization of adeno-iPS cells from different cell types. Oct4ind indicates Oct4ind allele present; GFP, GFP reporter gene present; ES, expression of ES cell markers (SSEA-1 and Oct4 or Sox2); TF, teratoma formation; PC, postnatal chimeras; GL, germline transmission. ND, not determined.

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The efficiency of deriving iPS-like cells from fetal liver cells, TTFs, and hepatocytes (see fig. S7 for an illustration of the derivation process from the different cell types) was extremely low, ranging from less than 0.0001% to 0.001% (Table 1). This frequency is lower than that obtained with integrating viruses (∼0.01 to 0.1%) and is probably due to the fact that many cells do not maintain viral expression long enough to trigger entry into a state sustained by endogenous pluripotency factors (24, 25). This conclusion is supported by qPCR analysis for adenoviral gene expression, which is gradually lost in dividing fibroblasts (fig. S8). It should be informative to test whether the low efficiency of adenoviral reprogramming can be increased by the use of chemical compounds as has been reported for retroviral reprogramming (2628).

DNA content analysis showed that 3 out of 13 (or about 23%) of the 13 adeno-iPS lines were tetraploid, which is not seen in iPS cells produced with retro- or lentiviral vectors (fig. S9 and Table 1). We speculate that adenoviral reprogramming either induces cell fusion or, alternatively, selects for rare tetraploid cells pre-existing in the starting cell populations. Indeed, it has been shown that the frequency of polyploid hepatocytes increases with age (29).

Our results demonstrate the generation of iPS cells without the use of integrating viruses by employing either a combination of adenoviruses and an inducible transgene or adenoviruses alone. Our work supports the claim that insertional mutagenesis is not required for in vitro reprogramming, and it provides a platform for studying the biology of iPS cells lacking viral integrations. For example, it should now be possible to assess if iPS cells and ES cells are equivalent at the molecular and functional levels. This comparison has not been possible so far because viral transgenes are expressed at low levels in iPS cells and their progeny, which may affect their molecular signatures, as well as their differentiation behavior and developmental potential. If human iPS cells can be generated without genome-integrating viruses, these cells may allow for the generation of safer patient-specific cells and thus could have important implications for cell therapy. Before translating these observations into a therapeutic setting, however, it will be important to assess if human iPS cells generated without viral integration are indeed as potent as human ES cells.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1162494/DC1

Materials and Methods

Figs. S1 to S10

Tables S1 to S3

References

References and Notes

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