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The Generation and Maintenance of Rat Induced Pluripotent Stem Cells

  • Tomoyuki YamaguchiEmail author
  • Sanae Hamanaka
  • Hiromitsu Nakauchi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1210)

Abstract

This chapter describes a newly developed method for generating and maintaining rat induced pluripotent stem cells (riPSCs). We first provide a detailed protocol for the generation of lentiviral vector carrying three reprogramming factors to produce high-quality riPSCs. This technique allows reprogramming of rat somatic cells to ground state with germ-line competence. Subsequently, we elaborate a detailed protocol for the generation of riPSCs from rat embryonic fibroblast (REF). Finally, the protocols for the optimal culture conditions of riPSCs and preparation of frozen stock are described. We also outline the advantages of generating riPSCs.

Key words

Rat induced pluripotent stem cells Lentiviral vector Dox inducible Germ-line competent GSK3 inhibitor MEK inhibitor 

References

  1. 1.
    Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676. doi: 10.1016/j.cell.2006.07.024, S0092-8674(06)00976-7 [pii]PubMedCrossRefGoogle Scholar
  2. 2.
    Okita K, Ichisaka T, Yamanaka S (2007) Generation of germLine-competent induced pluripotent stem cells. Nature 448(7151):313–317. doi: 10.1038/nature05934, nature05934 [pii]PubMedCrossRefGoogle Scholar
  3. 3.
    Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying QL (2008) GermLine competent embryonic stem cells derived from rat blastocysts. Cell 135(7):1299–1310. doi: 10.1016/j.cell.2008.12.006, S0092-8674(08)01567-5 [pii]PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying QL, Smith A (2008) Capture of authentic embryonic stem cells from rat blastocysts. Cell 135(7):1287–1298. doi: 10.1016/j.cell.2008.12.007, S0092-8674(08)01568-7 [pii]PubMedCrossRefGoogle Scholar
  5. 5.
    Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008) The ground state of embryonic stem cell self-renewal. Nature 453(7194):519–523. doi: 10.1038/nature06968, nature06968 [pii]PubMedCrossRefGoogle Scholar
  6. 6.
    Li W, Wei W, Zhu S, Zhu J, Shi Y, Lin T, Hao E, Hayek A, Deng H, Ding S (2009) Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4(1):16–19. doi: 10.1016/j.stem.2008.11.014, S1934-5909(08)00616-4 [pii]PubMedCrossRefGoogle Scholar
  7. 7.
    Hamanaka S, Yamaguchi T, Kobayashi T, Kato-Itoh M, Yamazaki S, Sato H, Umino A, Wakiyama Y, Arai M, Sanbo M, Hirabayashi M, Nakauchi H (2011) Generation of germLine-competent rat induced pluripotent stem cells. PLoS One 6(7):e22008. doi: 10.1371/journal.pone.0022008, PONE-D-11-06622 [pii]PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Yamaguchi T, Hamanaka S, Kamiya A, Okabe M, Kawarai M, Wakiyama Y, Umino A, Hayama T, Sato H, Lee YS, Kato-Itoh M, Masaki H, Kobayashi T, Yamazaki S, Nakauchi H (2012) Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming. PLoS One 7(7):e41007. doi: 10.1371/journal.pone.0041007, PONE-D-12-06326 [pii]PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Okabe M, Otsu M, Ahn DH, Kobayashi T, Morita Y, Wakiyama Y, Onodera M, Eto K, Ema H, Nakauchi H (2009) Definitive proof for direct reprogramming of hematopoietic cells to pluripotency. Blood 114(9):1764–1767. doi: 10.1182/blood-2009-02-203695, blood-2009-02-203695 [pii]PubMedCrossRefGoogle Scholar
  10. 10.
    Nishimura K, Sano M, Ohtaka M, Furuta B, Umemura Y, Nakajima Y, Ikehara Y, Kobayashi T, Segawa H, Takayasu S, Sato H, Motomura K, Uchida E, Kanayasu-Toyoda T, Asashima M, Nakauchi H, Yamaguchi T, Nakanishi M (2011) Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming. J Biol Chem 286(6):4760–4771. doi: 10.1074/jbc.M110.183780, M110.183780 [pii]PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Tomoyuki Yamaguchi
    • 1
    Email author
  • Sanae Hamanaka
    • 1
  • Hiromitsu Nakauchi
    • 1
  1. 1.Centre for Stem Cell Biology and Regenerative Medicine, Institute of Medical ScienceUniversity of TokyoMinato-kuJapan

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