Advertisement

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

  • Christian M. Nefzger
  • Sara Alaei
  • Jose M. PoloEmail author
Protocol
Part of the Methods in Molecular Biology book series (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).

Key words

Induced pluripotent stem cells Reprogrammable mouse model Reprogramming intermediates Cell surface marker Fluorescence-activated cell sorting 

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):663–676CrossRefPubMedGoogle Scholar
  2. 2.
    Stadtfeld M, Hochedlinger K (2010) Induced pluripotency: history, mechanisms, and applications. Genes Dev 24(20):2239–2263PubMedCentralCrossRefPubMedGoogle Scholar
  3. 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):861–872CrossRefPubMedGoogle Scholar
  4. 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):31–45CrossRefPubMedGoogle Scholar
  5. 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):402–406PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Ebert AD, Svendsen CN (2010) Human stem cells and drug screening: opportunities and challenges. Nat Rev Drug Discov 9(5):367–372CrossRefPubMedGoogle Scholar
  7. 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):58–62CrossRefPubMedGoogle Scholar
  8. 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):521–531CrossRefPubMedGoogle Scholar
  9. 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):68–73PubMedCentralCrossRefPubMedGoogle Scholar
  10. 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):64–77CrossRefPubMedGoogle Scholar
  11. 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):230–240PubMedCentralCrossRefPubMedGoogle Scholar
  12. 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):151–159PubMedCentralCrossRefPubMedGoogle Scholar
  13. 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):55–70CrossRefPubMedGoogle Scholar
  14. 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):1617–1632PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Stadtfeld M, Maherali N, Borkent M, Hochedlinger K (2009) A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nat Methods 7(1):53–55PubMedCentralCrossRefPubMedGoogle Scholar
  16. 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) 3854Google Scholar
  17. 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):1579–1592Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Christian M. Nefzger
    • 1
  • Sara Alaei
    • 1
  • Jose M. Polo
    • 1
    Email author
  1. 1.Department of Anatomy and Developmental BiologyAustralian Regenerative Medicine Institute, Monash UniversityClaytonAustralia

Personalised recommendations