Skip to main content
SpringerLink
Log in
Book cover

Cell Reprogramming pp 205–218Cite as

Isolation of Reprogramming Intermediates During Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts

  • Protocol

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1330))

Abstract

Mature cells of the body can be reprogrammed towards a pluripotent state by forced expression of the transcription factors Oct-4, Klf-4, Sox2, and C-Myc (OKSM) at very low efficiency. To study the reprogramming process in detail the rare intermediates of the reaction need to be separated from the bulk population. Using a genetically engineered reprogrammable mouse strain we describe how to isolate intermediates from reprogramming cultures of mouse embryonic fibroblasts via antibody labeling of cell surface markers and fluorescence-activated cell sorting (FACS).

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  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

    Article  CAS  PubMed  Google Scholar 

  2. Stadtfeld M, Hochedlinger K (2010) Induced pluripotency: history, mechanisms, and applications. Genes Dev 24(20):2239–2263

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872

    Article  CAS  PubMed  Google Scholar 

  4. Zhang J, Lian Q, Zhu G, Zhou F, Sui L, Tan C, Mutalif RA, Navasankari R, Zhang Y, Tse H-F (2011) A human iPSC model of Hutchinson Gilford Progeria reveals vascular smooth muscle and mesenchymal stem cell defects. Cell Stem Cell 8(1):31–45

    Article  CAS  PubMed  Google Scholar 

  5. Lee G, Papapetrou EP, Kim H, Chambers SM, Tomishima MJ, Fasano CA, Ganat YM, Menon J, Shimizu F, Viale A (2009) Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461(7262):402–406

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Ebert AD, Svendsen CN (2010) Human stem cells and drug screening: opportunities and challenges. Nat Rev Drug Discov 9(5):367–372

    Article  CAS  PubMed  Google Scholar 

  7. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Närvä E, Ng S, Sourour M, Hämäläinen R, Olsson C (2011) Copy number variation and selection during reprogramming to pluripotency. Nature 471(7336):58–62

    Article  CAS  PubMed  Google Scholar 

  8. Mayshar Y, Ben-David U, Lavon N, Biancotti J-C, Yakir B, Clark AT, Plath K, Lowry WE, Benvenisty N (2010) Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7(4):521–531

    Article  CAS  PubMed  Google Scholar 

  9. Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, Antosiewicz-Bourget J, O’Malley R, Castanon R, Klugman S (2011) Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471(7336):68–73

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Samavarchi-Tehrani P, Golipour A, David L, H-k S, Beyer TA, Datti A, Woltjen K, Nagy A, Wrana JL (2010) Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7(1):64–77

    Article  CAS  PubMed  Google Scholar 

  11. Stadtfeld M, Maherali N, Breault DT, Hochedlinger K (2008) Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2(3):230–240

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Brambrink T, Foreman R, Welstead GG, Lengner CJ, Wernig M, Suh H, Jaenisch R (2008) Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2(2):151–159

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R (2007) Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1(1):55–70

    Article  CAS  PubMed  Google Scholar 

  14. Polo JM, Anderssen E, Walsh RM, Schwarz BA, Nefzger CM, Lim SM, Borkent M, Apostolou E, Alaei S, Cloutier J (2012) A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 151(7):1617–1632

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Stadtfeld M, Maherali N, Borkent M, Hochedlinger K (2009) A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nat Methods 7(1):53–55

    Article  PubMed Central  PubMed  Google Scholar 

  16. Jozefczuk J, Drews K, Adjaye J (2012) Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. J Vis Exp (64) 3854

    Google Scholar 

  17. Hansson J, Rafiee MR, Reiland S, Polo JM, Gehring J, Okawa S, Huber W, Hochedlinger K, Krijgsveld J (2012) Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency. Cell Rep 2(6):1579–1592

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy and Developmental Biology, Australian Regenerative Medicine Institute, Monash University, Level 3, STRIP 1, Building 75, Wellington Road, Clayton, VIC, 3800, Australia

    Christian M. Nefzger, Sara Alaei & Jose M. Polo

Authors
  1. Christian M. Nefzger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sara Alaei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jose M. Polo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jose M. Polo .

Editor information

Editors and Affiliations

  1. Stem Cell and Genetic Engineering Group, Department of Materials Engineering Faculty of Engineering, Monash University, Clayton, Victoria, Australia

    Paul J. Verma

  2. Swinburne University of Technology, Hawthorn, Victoria, Australia

    Huseyin Sumer

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Nefzger, C.M., Alaei, S., Polo, J.M. (2015). Isolation of Reprogramming Intermediates During Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts. In: Verma, P., Sumer, H. (eds) Cell Reprogramming. Methods in Molecular Biology, vol 1330. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2848-4_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2848-4_17

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2847-7

  • Online ISBN: 978-1-4939-2848-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation