Generation of Induced Pluripotent Stem Cells with CytoTune, a Non-Integrating Sendai Virus

  • Pauline T. Lieu
  • Andrew Fontes
  • Mohan C. Vemuri
  • Chad C. MacArthur
Part of the Methods in Molecular Biology book series (MIMB, volume 997)


One of the major obstacles in generating induced pluripotent stem cells for research or downstream applications is the potential modifications of cellular genome as a result of using integrating viruses during reprogramming. Another major disadvantage of reprogramming cells with integrating vectors is that silencing and activation of transgenes are unpredictable, which may affect terminal differentiation potential and increase the risk of using iPSC-derived cells. Here we describe a protocol for the generation of induced pluripotent stem cells using a non-integrating RNA virus, Sendai virus, to efficiently generate transgene-free iPSCs starting with different cell types as well as in feeder-free conditions.

Key words

Induced pluripotent stem cells Non-integrating Sendai virus High efficiency Human cells Reprogramming Feeder free 



This work was supported by Life Technologies Corporation. The products within this publication are for Research Use Only, not intended for animal or human therapeutic or diagnostic use.


  1. 1.
    Takeda A, Igarashi H, Kawada M, Tsukamoto T, Yamamoto H, Inoue M, Iida A, Shu T, Hasegawa M, Matano T (2008) Evaluation of the immunogenicity of replication-competent V-knocked-out and replication-defective F-deleted Sendai virus vector-based vaccines in macaques. Vaccine 26:6839–6843PubMedCrossRefGoogle Scholar
  2. 2.
    Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318–324PubMedCrossRefGoogle Scholar
  3. 3.
    Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920PubMedCrossRefGoogle Scholar
  4. 4.
    Park IH, Lerou PH, Zhao R, Huo H, Daley GQ (2008) Generation of human-induced pluripotent stem cells. Nat Protoc 3: 1180–1186PubMedCrossRefGoogle Scholar
  5. 5.
    Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321:1218–1221PubMedCrossRefGoogle Scholar
  6. 6.
    Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN (2009) Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457: 277–280PubMedCrossRefGoogle Scholar
  7. 7.
    Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, Hargus G, Blak A, Cooper O, Mitalipova M, Isacson O, Jaenisch R (2009) Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136:964–977PubMedCrossRefGoogle Scholar
  8. 8.
    Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448:313–317PubMedCrossRefGoogle Scholar
  9. 9.
    Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8:409–412PubMedCrossRefGoogle Scholar
  10. 10.
    Muller LU, Daley GQ, Williams DA (2009) Upping the ante: recent advances in direct reprogramming. Mol Ther 17:947–953PubMedCrossRefGoogle Scholar
  11. 11.
    Li HO, Zhu YF, Asakawa M, Kuma H, Hirata T, Ueda Y, Lee YS, Fukumura M, Iida A, Kato A, Nagai Y, Hasegawa M (2000) A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression. J Virol 74:6564–6569PubMedCrossRefGoogle Scholar
  12. 12.
    Ikeda Y, Yonemitsu Y, Sakamoto T, Ishibashi T, Ueno H, Kato A, Nagai Y, Fukumura M, Inomata H, Hasegawa M, Sueishi K (2002) Recombinant Sendai virus-mediated gene transfer into adult rat retinal tissue: efficient gene transfer by brief exposure. Exp Eye Res 75:39–48PubMedCrossRefGoogle Scholar
  13. 13.
    Masaki I, Yonemitsu Y, Komori K, Ueno H, Nakashima Y, Nakagawa K, Fukumura M, Kato A, Hasan MK, Nagai Y, Sugimachi K, Hasegawa M, Sueishi K (2001) Recombinant Sendai virus-mediated gene transfer to ­vasculature: a new class of efficient gene transfer vector to the vascular system. FASEB J 15:1294–1296PubMedGoogle Scholar
  14. 14.
    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. Proc Jpn Acad 85:348–362CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Pauline T. Lieu
    • 1
  • Andrew Fontes
    • 1
  • Mohan C. Vemuri
    • 2
  • Chad C. MacArthur
    • 1
  1. 1.Primary and Stem Cell SystemsLife TechnologiesCarlsbadUSA
  2. 2.Primary and Stem Cell SystemsLife TechnologiesFrederickUSA

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