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Stem Cell Reviews and Reports

, Volume 9, Issue 4, pp 435–450 | Cite as

Generation of Mouse and Human Induced Pluripotent Stem Cells (iPSC) from Primary Somatic Cells

  • I. M. Lorenzo
  • A. Fleischer
  • D. Bachiller
Article

Abstract

Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663–676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotency-associated genes Okita et al. (Science 322(5903):949–953, 2008), lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543–549, 2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85(8):348–362, 2009), removal of the reprogramming vectors by ‘piggyBac’ transposition Woltjen et al. (Nature 458(7239):766–770, 2009); Kaji et al. (Nature 458(7239):771–775, 2009), Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964–977, 2009), episomal vectors Yu et al. (Science 324(5928):797–801, 2009), cell-penetrating reprogramming proteins Zhou et al. (Stem Cells 4(5):381–384, 2009), mammalian artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011) synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012), miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376–388, 2009); however, although some of these methods are commercially available, in general they still need to attain the reproducibility and reprogramming efficiency required for routine applications Mochiduki and Okita (Biotechnol Journal 7(6):789–797, 2012). Herein we explain, in four detailed protocols, the isolation of mouse and human somatic cells and their reprogramming into iPSC. All-encompassing instructions, not previously published in a single document, are provided for mouse and human iPSC colony isolation and derivation. Although mouse and human iPSC share similarities in the cellular reprogramming process and culture, both cell types need to be handled differently.

Keywords

Induced pluripotent stem cells Cellular reprogramming Primary keratinocytes Primary mouse embryonic fibroblasts Retrovirus Feeder cells Cell derivation 

Notes

Acknowledgments

This work was supported by the MICINN-JDC and MICINN PLE2009-0091 and IPT-2011-1402-900000 grants. We are grateful to J.C. Izpisúa-Belmonte and T. Aasen from the Center of Regenerative Medicine in Barcelona (CRMB) for their help in establishing iPSC technology in our lab, as well as for the gift of human OKSM retroviral plasmids and Ecotropic Phoenix cells. We also thank S. Yamanaka for the gift of mouse OKSM retroviral plasmids, and M.V. Camarasa for technical advise on stem cell culture. We also thank the Balearic Islands University Institute for Biomedical Research (IUNICS) for the use of their facilities.

Conflict of interest

The authors declare no potential conflicts of interest.

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Caubet-Cimera Foundation, Centre for Advanced Respiratory MedicineBunyolaSpain
  2. 2.Consejo Superior de Investigaciones Científicas (CSIC)MallorcaSpain

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