Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced pluripotent stem cells generated without viral integration. Science 2008;322:945–949. (Reprinted with permission.)
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. Here, we generate mouse induced pluripotent stem (iPS) cells from fibroblasts and liver cells by using non-integrating 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 germline, 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 conversion of differentiated somatic cells into pluripotent stem cells, also called “nuclear reprogramming”, was known for more than a decade to happen after the transfer of a somatic nucleus into an enucleated oocyte. Recently, the pioneering work of Shinya Yamanaka1 demonstrated that a retrovirus-mediated overexpression of four genes (Oct4 [octamer-binding transcription factor 4, POU5F1], Klf4 [Kruppel-like factor 4], Sox2 [sex-determining region Y box 2], and c-Myc) in murine fibroblasts is sufficient to mimic nuclear reprogramming to an embryonic-like state, resulting in “induced pluripotent stem cells” (iPS cells). This work was further refined2 and reproduced by others3, 4 and is widely accepted to be an ideal technique to generate pluripotent stem cells from nonembryonic cell sources. In the meantime, the generation of iPS cells was also reported for human cells, using adult fibroblasts or mesenchymal stem cells as starting populations.5–7
Among the numerous issues of iPS cell generation that have been addressed and are currently investigated in various laboratories, some are of particular importance for future applications: First, potential side effects of the retrovirus-mediated gene transfer, such as insertional mutagenesis, need to be reduced or eliminated. Second, an increased tumor formation rate of iPS cell–derived tissues in chimeric mice is reported. Third, the most appropriate starting cell population for generating iPS cells needs to be defined. In an earlier report, the group of Yamanaka demonstrated that primary hepatocytes isolated from the same F-box protein 15 (Fbx15)-reporter mouse model as used in the pioneering work of 2006 are even more efficiently reprogrammed to iPS cells than fibroblasts.8 Furthermore, in hepatocyte-derived iPS cells no common retroviral insertion sites could be demonstrated, casting doubt on insertional mutagenesis as one key event during iPS cell generation.
In a recent issue of science, Matthias Stadtfeld et al. reported on iPS cell generation using nonintegrating adenovirus-mediated gene transfer.9 Initially, the authors tried to reprogram mouse tail-tip fibroblasts to an embryonic stem (ES) cell–like state by adenoviral delivery of the four transcription factors Oct4, Sox2, c-Myc, and Klf4. However, this attempt was unsuccessful, probably owing to a low transduction efficiency and rapid dilution of the viral transgenes. Efficient reprogramming of somatic cells seems to depend on active expression of all four transgenes at a certain constant level over a longer period of time. To lower the number of factors to be transduced with adenoviruses, Stadtfeld et al. substituted viral Oct4 expression by a doxycycline-inducible Oct4 allele driven by a reverse-tetracycline-dependent transactivator present in the ROSA26 locus (Oct4IND), as described.10 Moreover, it was reported that liver cells require less viral transgene integrations than fibroblasts for efficient reprogramming.8 Therefore, Stadtfeld et al. infected Oct4IND fetal mouse liver cells with adenoviruses expressing Sox2, Klf4, and c-Myc at multiplicities of infection (MOI, number of active viral particles per cell) of 20-50, which led to an estimated infection efficiency of 20%-30% for cells expressing all three adenoviral transgenes. Thereafter, Oct4IND-infected fetal liver cells were cultured in the presence of doxycycline for 24-30 days. Nine iPS cell–like colonies were isolated and could be expanded into ES cell–like lines in which addition of doxycyclin for induction of transgenic Oct4 expression was no longer required. On the basis of these results, Stadtfeld et al. further refined their method to reprogram primary adult hepatocytes isolated from mice carrying the Oct4-GFP and ROSA26-rRTA alleles, but lacking the Oct4-inducible allele. MOIs of 1-4 were found to be sufficient to reprogram hepatocytes, because these cells are highly permissive for adenoviral infection. Approximately 50%-60% of all cells were positive for the expression of all four transgenes, which gave rise to three colonies positive for green fluorescent protein that could be expanded to stable ES cell–like cell lines. Southern blot as well as polymerase chain reaction analysis of genomic DNA from adeno-iPS cells confirmed the absence of integrated adenoviral genes in all iPS cell lines tested. Thus, more than 2 years after the establishment of the iPS cell technology by Shinya Yamanaka, these newly generated adeno-iPS cells are the first reported reprogrammed pluripotent stem cells with evidence of a complete lack of viral transgene integration. However, depending on the somatic cell type used in this assay, the efficiency of iPS cell generation was in the range of 0.0001%-0.0018%, which is around three magnitudes lower when compared to the retroviral transduction protocol (0.1%) and in the same range as using improved nonviral transfection methods in fetal fibroblasts as described by Okita et al. in the same issue of science.111
Taken together, various iPS cell lines were established from murine fetal liver cells and adult hepatocytes. One fetal liver cell–derived and two hepatocyte-derived iPS cell lines were tetraploid, which the authors omitted in the characterization for pluripotency and further experiments. All of the remaining iPS cell lines showed gene expression profiles that were indistinguishable from ES cells, and pluripotency was demonstrated in six individual iPS cell lines by teratoma formation. Two fetal liver cell–derived as well as the diploid hepatocyte-derived iPS cell lines were tested positive for their ability to contribute to postnatal chimeras after blastocyst injection. However, the ultimate proof of germline contribution was determined only for one fetal liver cell–derived iPS cell line. Nevertheless, with this elegant study the authors address the feasibility of nuclear reprogramming and iPS cell generation without permanent genetic alterations, which is important in circumventing potential tumorigenic risks. Furthermore, the lack of persistent genetic alterations allows a more accurate comparison of iPS and ES cells at the molecular and functional levels, which should be a further step toward assessing the tenability of iPS cell employment in clinical studies. Last but not least, because gene transfer–mediated alterations affecting the experimental read-out can be widely excluded, such iPS cell generation might be a preferred method for studying disease-specific or patient-specific iPS cells for new therapeutic approaches or drug screenings.