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Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach

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Abstract

Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.

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References

  1. Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–676.

    Article  CAS  Google Scholar 

  2. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872.

    Article  CAS  Google Scholar 

  3. Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I., et al. (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science, 324, 797–801.

    Article  CAS  Google Scholar 

  4. Wang, W., Yang, J., Liu, H., Lu, D., Chen, X., Zenonos, Z., et al. (2011). Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1. Proceedings of the National Academy of Sciences USA, 108, 18283–18288.

    Article  CAS  Google Scholar 

  5. Bayart, E., & Cohen-Haguenauer, O. (2013). Technological overview of iPS induction from human adult somatic cells. Current Gene Therapy, 13, 73–92.

    Article  CAS  Google Scholar 

  6. Zhou, Y. Y., & Zeng, F. (2013). Integration-free methods for generating induced pluripotent stem cells. Genomics Proteomics Bioinformatics, 11, 284–287.

    Article  CAS  Google Scholar 

  7. Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., et al. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 448, 318–324.

    CAS  Google Scholar 

  8. Okita, K., Ichisaka, T., & Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature, 448, 313–317.

    CAS  Google Scholar 

  9. Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., et al. (2008). Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science, 321, 699–702.

    CAS  Google Scholar 

  10. Hamilton B, Feng Q, Ye M, Welstead GG (2009) Generation of induced pluripotent stem cells by reprogramming mouse embryonic fibroblasts with a four transcription factor, doxycycline inducible lentiviral transduction system. J Vis Exp. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19915522.

  11. Sommer, C. A., Stadtfeld, M., Murphy, G. J., Hochedlinger, K., Kotton, D. N., & Mostoslavsky, G. (2009). Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells, 27, 543–549.

    CAS  Google Scholar 

  12. Winkler, T., Cantilena, A., Metais, J. Y., Xu, X., Nguyen, A. D., Borate, B., et al. (2010). No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells. Stem Cells, 28, 687–694.

    CAS  Google Scholar 

  13. Thomson, M., Liu, S. J., Zou, L. N., Smith, Z., Meissner, A., & Ramanathan, S. (2011). Pluripotency factors in embryonic stem cells regulate differentiation into germ layers. Cell, 145, 875–889.

    CAS  Google Scholar 

  14. Somers, A., Jean, J.-C., Sommer, C. A., Omari, A., Ford, C. C., Mills, J. A., et al. (2010). Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells Dayt Ohio, 28, 1728–1740.

    CAS  Google Scholar 

  15. Papapetrou, E. P., Lee, G., Malani, N., Setty, M., Riviere, I., Tirunagari, L. M. S., et al. (2011). Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells. Nature Biotechnology, 29, 73–78.

    CAS  Google Scholar 

  16. Loh, Y.-H., Yang, J. C., De Los, Angeles. A., Guo, C., Cherry, A., Rossi, D. J., et al. (2012). Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA. Current Protocols in Stem Cell Biology Chapter, 4(Unit4A), 5.

    Google Scholar 

  17. Sommer, C. A., Sommer, A. G., Longmire, T. A., Christodoulou, C., Thomas, D. D., Gostissa, M., et al. (2010). Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector. Stem Cells Dayt Ohio, 28, 64–74.

    CAS  Google Scholar 

  18. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., & Yamanaka, S. (2008). Generation of mouse induced pluripotent stem cells without viral vectors. Science, 322, 949–953.

    CAS  Google Scholar 

  19. Zhou, W., & Freed, C. R. (2009). Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells, 27, 2667–2674.

    CAS  Google Scholar 

  20. Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., & Hasegawa, M. (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences, 85, 348–362.

    CAS  Google Scholar 

  21. Woltjen, K., Michael, I. P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., et al. (2009). piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 458, 766–770.

    CAS  Google Scholar 

  22. Warren, L., Manos, P. D., Ahfeldt, T., Loh, Y. H., Li, H., Lau, F., et al. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 7, 618–630.

    CAS  Google Scholar 

  23. Zhou, H., Wu, S., Joo, J. Y., Zhu, S., Han, D. W., Lin, T., et al. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 4, 381–384.

    CAS  Google Scholar 

  24. Hou, P., Li, Y., Zhang, X., Liu, C., Guan, J., Li, H., et al. (2013). Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science, 341, 651–654.

    CAS  Google Scholar 

  25. Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S., & Calos, M. P. (2001). Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Molecular and Cellular Biology, 21, 3926–3934.

    CAS  Google Scholar 

  26. Nagy, A. (2000). Cre recombinase: The universal reagent for genome tailoring. Genesis, 26, 99–109.

    CAS  Google Scholar 

  27. Zhao, S., Maxwell, S., Jimenez-Beristain, A., Vives, J., Kuehner, E., Zhao, J., et al. (2004). Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. European Journal of Neuroscience, 19, 1133–1140.

    Google Scholar 

  28. Lee, S. H., Lumelsky, N., Studer, L., Auerbach, J. M., & McKay, R. D. (2000). Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nature Biotechnology, 18, 675–679.

    CAS  Google Scholar 

  29. Baron, U., Freundlieb, S., Gossen, M., & Bujard, H. (1995). Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Research, 23, 3605–3606.

    CAS  Google Scholar 

  30. Szymczak, A. L., Workman, C. J., Wang, Y., Vignali, K. M., Dilioglou, S., Vanin, E. F., et al. (2004). Correction of multi-gene deficiency in vivo using a single “self-cleaving” 2A peptide-based retroviral vector. Nature Biotechnology, 22, 589–594.

    CAS  Google Scholar 

  31. Szymczak, A. L., & Vignali, D. A. (2005). Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opinon Biological Therapy, 5, 627–638.

    CAS  Google Scholar 

  32. Choi, T., Huang, M., Gorman, C., & Jaenisch, R. (1991). A generic intron increases gene expression in transgenic mice. Molecular and Cellular Biology, 11, 3070–3074.

    CAS  Google Scholar 

  33. Freundlieb, S., Schirra-Muller, C., & Bujard, H. (1999). A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. The Journal of Gene Medicine, 1, 4–12.

    CAS  Google Scholar 

  34. Bornkamm, G. W., Berens, C., Kuklik-Roos, C., Bechet, J. M., Laux, G., Bachl, J., et al. (2005). Stringent doxycycline-dependent control of gene activities using an episomal one-vector system. Nucleic Acids Research, 33, e137.

    Google Scholar 

  35. Niwa, H., Yamamura, K., & Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108, 193–199.

    CAS  Google Scholar 

  36. Groth, A. C., Olivares, E. C., Thyagarajan, B., & Calos, M. P. (2000). A phage integrase directs efficient site-specific integration in human cells. Proceedings of the National Academy of Sciences USA, 97, 5995–6000.

    CAS  Google Scholar 

  37. Raymond, C. S., & Soriano, P. (2007). High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS ONE, 2, e162.

    Google Scholar 

  38. Hitz, C., Steuber-Buchberger, P., Delic, S., Wurst, W., & Kuhn, R. (2009). Generation of shRNA transgenic mice. Methods in Molecular Biology, 530, 101–129.

    CAS  Google Scholar 

  39. Bishop, C. E., & Hatat, D. (1987). Molecular cloning and sequence analysis of a mouse Y chromosome RNA transcript expressed in the testis. Nucleic Acids Research, 15, 2959–2969.

    CAS  Google Scholar 

  40. Uren, A. G., Mikkers, H., Kool, J., van der Weyden, L., Lund, A. H., Wilson, C. H., et al. (2009). A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nature Protocols, 4, 789–798.

    CAS  Google Scholar 

  41. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402–408.

    CAS  Google Scholar 

  42. Pollack, J. R., Sorlie, T., Perou, C. M., Rees, C. A., Jeffrey, S. S., Lonning, P. E., et al. (2002). Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumours. Proceedings of the National Academy of Sciences USA, 99, 12963–12968.

    CAS  Google Scholar 

  43. Frigola, J., Song, J., Stirzaker, C., Hinshelwood, R. A., Peinado, M. A., & Clark, S. J. (2006). Epigenetic remodelling in colorectal cancer results in coordinate gene suppression across an entire chromosome band. Nature Genetics, 38, 540–549.

    CAS  Google Scholar 

  44. Skylaki, S., & Tomlinson, S. R. (2012). Recurrent transcriptional clusters in the genome of mouse pluripotent stem cells. Nucleic Acids Research, 40, e153.

    CAS  Google Scholar 

  45. Urlinger, S., Baron, U., Thellmann, M., Hasan, M. T., Bujard, H., & Hillen, W. (2000). Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Proceedings of the National Academy of Sciences USA, 97, 7963–7968.

    CAS  Google Scholar 

  46. Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S., & Calos, M. P. (2001). Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Molecular and Cellular Biology, 21, 3926–3934.

    CAS  Google Scholar 

  47. Chalberg, T. W., Portlock, J. L., Olivares, E. C., Thyagarajan, B., Kirby, P. J., Hillman, R. T., et al. (2006). Integration specificity of phage phiC31 integrase in the human genome. Journal of Molecular Biology, 357, 28–48.

    CAS  Google Scholar 

  48. Esteban, M. A., Wang, T., Qin, B., Yang, J., Qin, D., Cai, J., et al. (2010). Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 6, 71–79.

    CAS  Google Scholar 

  49. Teng, H. F., Kuo, Y. L., Loo, M. R., Li, C. L., Chu, T. W., Suo, H., et al. (2010). Valproic acid enhances Oct4 promoter activity in myogenic cells. Journal of Cellular Biochemistry, 110, 995–1004.

    CAS  Google Scholar 

  50. Ye, L., Chang, J. C., Lin, C., Qi, Z., Yu, J., & Kan, Y. W. (2010). Generation of induced pluripotent stem cells using site-specific integration with phage integrase. Proceedings of the National Academy of Sciences USA, 107, 19467–19472.

    CAS  Google Scholar 

  51. Anokye-Danso, F., Trivedi, C. M., Juhr, D., Gupta, M., Cui, Z., Tian, Y., et al. (2011). Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell, 8, 376–388.

    CAS  Google Scholar 

  52. Mayshar, Y., Ben-David, U., Lavon, N., Biancotti, J. C., Yakir, B., Clark, A. T., et al. (2010). Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell, 7, 521–531.

    CAS  Google Scholar 

  53. Smidt, M. P., van Schaick, H. S., Lanctôt, C., Tremblay, J. J., Cox, J. J., van der Kleij, A. A., et al. (1997). A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proceedings of the National Academy of Sciences USA, 94, 13305–13310.

    CAS  Google Scholar 

  54. Merkle, F. T., & Eggan, K. (2013). Modelling human disease with pluripotent stem cells: from genome association to function. Cell Stem Cell, 12, 656–668.

    CAS  Google Scholar 

  55. Engle, S. J., & Puppala, D. (2013). Integrating human pluripotent stem cells into drug development. Cell Stem Cell, 12, 669–677.

    CAS  Google Scholar 

  56. Yu, D. X., Marchetto, M. C., & Gage, F. H. (2013). Therapeutic translation of iPSCs for treating neurological disease. Cell Stem Cell, 12, 678–688.

    CAS  Google Scholar 

  57. Lan, F., Liu, J., Narsinh, K. H., Hu, S., Han, L., Lee, A. S., et al. (2012). Safe genetic modification of cardiac stem cells using a site-specific integration technique. Circulation, 126, S20–S28.

    CAS  Google Scholar 

  58. Chavez, C. L., & Calos, M. P. (2011). Therapeutic applications of the ΦC31 integrase system. Current Gene Therapy, 11, 375–381.

    CAS  Google Scholar 

  59. Karow, M., & Calos, M. P. (2011). The therapeutic potential of ΦC31 integrase as a gene therapy system. Expert Opinion on Biological Theraphy, 11, 1287–1296.

    CAS  Google Scholar 

  60. Karow, M., Chavez, C. L., Farruggio, A. P., Geisinger, J. M., Keravala, A., Jung, W. E., et al. (2011). Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA. Stem Cells Dayt Ohio, 29, 1696–1704.

    CAS  Google Scholar 

  61. Collins, F. S., Rossant, J., & Wurst, W. (2007). A mouse for all reasons. Cell, 128, 9–13.

    CAS  Google Scholar 

  62. Lee, A. Y.-F., & Lloyd, K. C. K. (2011). Rederivation of transgenic mice from iPS cells derived from frozen tissue. Transgenic Research, 20, 167–175.

    CAS  Google Scholar 

  63. Merkl, C., Saalfrank, A., Riesen, N., Kühn, R., Pertek, A., Eser, S., et al. (2013). Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector. PLoS ONE, 8, e55170.

    CAS  Google Scholar 

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Acknowledgments

We thank R. Kneuttinger, C. Arndt, A. Tasdemir, S. Weidemann, Anke Bettenbrock and H. Krause for excellent technical assistance and Meng Li (Imperial College London) for Pitx3GFP mice. This work was supported by the Bundesministerium für Bildung und Forschung program on ‘Gewinnung pluri-bzw. multipotenter Stammzellen’ (FKZ 01 GN 0806) and the DiGtoP consortium (FKZ01GS0858), the Deutsche Forschungsgemeinschaft within the Munich Cluster for Systems Neurology (EXC 1010, SyNergy), the Helmholtz Alliance HelMA and by the European Commission (EUCOMMtools, HEALTH-F4-2010-261492).

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  1. Anna Pertek

    Present address: Institute of Stem Cell Research, Helmholtz Zentrum München, 85764, Munich, Germany

  2. Stavroula Skylaki

    Present address: Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland

Authors and Affiliations

  1. Institute of Developmental Genetics, Helmholtz Zentrum München, 85764, Munich, Germany

    Anna Pertek, Florian Meier, Wolfgang Wurst, Nilima Prakash & Ralf Kühn

  2. Institute of Experimental Genetics, Helmholtz Zentrum München, 85764, Munich, Germany

    Martin Irmler & Johannes Beckers

  3. Chair of Developmental Genetics, Technical University Munich, 85350, Freising, Weihenstephan, Germany

    Florian Meier, Wolfgang Wurst, Nilima Prakash & Ralf Kühn

  4. Chair of Experimental Genetics, Technical University Munich, 85350, Freising, Weihenstephan, Germany

    Johannes Beckers

  5. Deutsches Zentrum für Neurodegenerative Erkrankungen e. V, 80336, Munich, Germany

    Wolfgang Wurst

  6. Max-Planck-Institute of Psychiatry, 80804, Munich, Germany

    Wolfgang Wurst

  7. Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-Universität München, 80336, Munich, Germany

    Wolfgang Wurst

  8. Helmholtz Center Munich, Ingolstädter Landstr. 1, 85764, Munich, Germany

    Ralf Kühn

  9. Institute of Stem Cell Research, Helmholtz Zentrum München, 85764, Munich, Germany

    Stavroula Skylaki & Max Endele

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  1. Anna Pertek
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Correspondence to Ralf Kühn.

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Pertek, A., Meier, F., Irmler, M. et al. Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach. Mol Biotechnol 56, 697–713 (2014). https://doi.org/10.1007/s12033-014-9748-y

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