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Copyright © 2007 Cell Press. All rights reserved. Cell, Vol 131, 834-835, 30 November 2007

Preview Induction of Pluripotency: From Mouse to Human Holm Zaehres1 and Hans R. Schöler1 ,

1 Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Münster, NRW 48149, Germany

Corresponding author Hans R. Schöler [email protected]

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In this issue of Cell, Takahashi et al., 2007 transfer their seminal work on somatic cell reprogramming from the mouse to human. By overexpressing the transcription factor quartet of Oct4, Sox2, Klf4, and c-Myc in adult human fibroblasts, they successfully isolate human pluripotent stem cells that resemble human embryonic stem cells by all measured criteria. This is a significant turning point in nuclear reprogramming research with broad implications for generating patient-specific pluripotent stem cells for research and therapeutic applications.

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This year's three Physiology or Medicine Nobel Laureates—Martin Evans, Mario Capecchi, and Oliver Smithies—will be honored in Stockholm in 10 days time for their discovery of DNA recombination and the development of mouse embryonic stem (ES) cell technology. It was Martin Evans who discovered how to make mouse ES cells, enabling any genetic alteration to be transferred to the germline and hence to the next generation (Evans et al., 1981 , Martin, 1981 ). Before this breakthrough, researchers studied mouse embryonal carcinoma cells derived from tumors, which could form every mouse cell lineage except the germline. Combining DNA recombination and mouse ES cell technology revolutionized an entire field of research, forming the basis for studying and understanding the roles of numerous genes in embryonic development, adult physiology, disease, and aging. To date, more than 500 mouse models of human disorders have been generated. Now, with the study by Takahashi et al., 2007 published in this issue of Cell, another important revolution is taking place. Last summer, Takahashi et al., 2006 stunned the scientific community with their study showing molecular reprogramming of mouse somatic cells into induced pluripotent stem (iPS) cells using just four factors: Oct4, Sox2, Klf4, and c-Myc. Their elegant but demanding approach of screening for a cocktail of factors that could reprogram mouse fibroblasts starting from 24 candidate genes paid off with their detailed description of iPS cells, which are almost indistinguishable from mouse ES cells. As with all scientific discoveries, these exciting findings had to be reproduced. Several studies published

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this year not only reproduced but also extended the Takahashi and Yamanaka findings by demonstrating the pluripotency and differentiation potential of mouse iPS cells in rigorous developmental assays (Maherali et al., 2007 , Okita et al., 2007 , Wernig et al., 2007 ). In their new study, Takahashi, Yamanaka, and their colleagues (Takahashi et al., 2007 ) now translate their remarkable findings from mouse to human (see Figure 1). They selected adult human dermal fibroblasts and two other human fibroblast populations (from synovial tissue and neonatal foreskin) from different human donors as their reprogramming target cell populations. They then transduced the human fibroblast cultures with retroviral vectors carrying transgenes for the human versions of Oct4, Sox2, Klf4, and c-Myc and cultured the cells under human ES cell culture conditions. Thirty days after transduction, the culture plates were covered with human ES cell-like iPS colonies (among other colonies), which could be further propagated and expanded. The retroviral vectors enabled sil encing of all four transgenes after human iPS formation (as found in the mouse system) indicating that the iPS cells are fully reprogrammed and no longer depend on transgene expression.

Figure 1. Transcription Factor-Induced Pluripotency Adult fibroblasts from human donors were exposed to retroviral vectors expressing a cocktail of four transgenes encoding the human factors hOct4, hSox2, hKlf4, and hc-Myc (Takahashi et al., 2007 ). Thirty days after transduction and further cultivation under human ES cell growth conditions, human induced pluripotent stem (iPS) cell colonies (among others) that could be propagated and expanded further were isolated. Comparative analysis of human iPS cells and human ES cells using assays for morphology, surface-marker expression, gene expression profiling, epigenetic status, and in vitro and in vivo differentiation potential revealed a remarkable degree of similarity between these two pluripotent stem cell types. View larger version: [In this window] [In new window]

Unlike the mouse study, human iPS cells were generated without any genetic selection procedures. Given the lower mitotic index of human ES cells, it is not surprising that the generation of human iPS cells takes notably longer than in the mouse system. The authors subjected their human iPS cells to a panel of assays to compare them with human ES cells. These assays included morphological studies, surface-marker expression, epigenetic status, formation of embryoid bodies in vitro, directed differentiation into neural cells and beating cardiomyocytes (according to human ES cell differentiation protocols), and finally teratoma formation in vivo. DNA microarray analysis revealed the remarkable degree of similarity between the global gene expression patterns of human iPS cells and human ES cells. Notably, genomic DNA analysis as well as analysis of short tandem repeats demonstrated the genetic origin of independent human iPS clones from their parental fibroblast populations. The derivation of mouse and then human ES cells (Thomson et al., 1998 ) as the gold standard of pluripotent stem cell populations has necessarily led to emphasis on differences in the regulation of self-renewal between mouse and human ES cells. For example, human ES cells depend on bFGF for self-renewal, whereas their mouse counterparts depend on the Lif/Stat3 pathway; BMP is involved in mouse ES cell self-renewal, whereas in human ES cells it induces differentiation. Extrinsic factors and signals for maintaining pluripotency may differ between mouse and human. However, the ability to translate somatic cell reprogramming from mouse to human using the same transcription factor quartet further emphasizes the conserved nature of the Oct4/Sox2 transcription factor network that controls self-renewal of mouse and human ES cells (Boyer et al., 2005 ). Given that Klf4 and c-Myc are



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chromatin modifiers and can immortalize cells, one might be able to find other factors or small molecules that could replace these two factors in the cocktail (Yamanaka, 2007 ). In these studies, the possibility of retroviral insertional mutagenesis, resulting in the activation of other genes contributing to reprogramming, cannot be excluded, providing an opportunity to potentially identify new reprogramming factors beyond the current quartet. Also, taking a broader screening approach for reprogramming human fibroblasts (as Takahashi and Yamanaka did for their mouse study) might yield other combinations of reprogramming factors. Direct reprogramming of somatic cells to a pluripotent state, thus reversing the developmental arrow of time, is considered by some to be the “holy grail” of stem cell research. Once the results in human cells are confirmed, these advances will enable the creation of patient-specific stem cell lines to study different disease mechanisms in the laboratory. Such cellular models also have the potential to dramatically increase the efficiency of drug discovery and to provide valuable tools for toxicology testing. Furthermore, this reprogramming system could make the idea of customized patient-specific screening and therapy both possible and economically feasible. Finally, the work will have a powerful impact on the intense debate regarding the moral, religious, and political aspects of ES cell research. However, a big mistake now would be to consider human ES cells obsolete. There are still many hurdles to overcome before we fully understand pluripotency and before we have human iPS cells in hand that are suitable for therapeutic application. For example, a significant proportion of mice derived from mouse iPS cells develop tumors due to reactivation of the c-Myc retrovirus (Okita et al., 2007 ) compared to mice derived from ES cells, which are normal. The search is now on to find a way to reprogram somatic cells without retroviruses and maybe even using a cocktail of small molecules. Given this, it should be emphasized that human ES cell research is more important than ever for it will shed light on how iPS cells can best be maintained in their pluripotent state and how they can be induced to differentiate into the cell lineage of interest. The field of nuclear reprogramming has come a long way from the initial nuclear transplantation studies in frogs 50 years ago, to the birth of Dolly, the first mammal cloned from adult somatic cells (Wilmut et al., 1997 ), to the fallout from the fabricated human nuclear transfer experiments of several years ago, to the landmark studies of Takahashi, Yamanaka, and their colleagues, first in mice and now in humans. References Summary

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References

Boyer, L.A., Lee, T.I., Cole, M.F., Johnstone, S.E., Levine, S.S., Zucker, J.P., Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G. et al. (2005). Cell 122, 947-956. [Medline] [Summary] [Full Text] Evans, M.J., and Kaufman, M.H. (1981). Nature 292, 154-156. [Medline]

Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachenko, R., Tchieu, J., Jaenisch, R. et al. (2007). Cell Stem Cell 1, 55-70. [Medline] Martin, G.R. (1981). Proc. Natl. Acad. Sci. USA 78, 7634-7638. [Medline] Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317. [Medline] Takahashi, K., and Yamanaka, S. (2006). Cell 126, 663-676. [Medline] [Summary] [Full Text] Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Cell, this issue.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Science 282, 1145-1147. [Medline]



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Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R. (2007). Nature 448, 318-324. [Medline] Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., and Campbell, K.H. (1997). Nature 385, 810-813. [Medline] Yamanaka, S. (2007). Cell Stem Cell 1, 39-49. [Medline]

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