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Embryonic Stem Cell Protocols pp 25–40Cite as

Resolving Heterogeneity: Fluorescence-Activated Cell Sorting of Dynamic Cell Populations from Feeder-Free Mouse Embryonic Stem Cell Culture

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1341))

Abstract

Embryonic stem cell (ESC) culture comprises a mixture of cells that are primed to differentiate into different lineages. In conditions where ESCs self-renew, these primed populations continuously interconvert and consequently show highly dynamic coordinated changes in their expression of different sets of pluripotency and differentiation markers. It has become increasingly apparent that this transcriptional heterogeneity is an important characteristic of ESC culture. By sorting for specific populations of ESCs it is possible to enrich for cells with a capacity to colonize the embryo proper or the extra-embryonic lineages such as the descendents of the primitive endoderm or trophoblast. Here, we describe a method of isolating specific sub-sets of ESCs from the pluripotent cells present in in vitro ESC culture using SSEA1 antibody staining in combination with reporter lines and fluorescence activated cell sorting (FACS).

An erratum to this chapter is available at 10.1007/7651_2015_254

An erratum to this chapter can be found at http://dx.doi.org/10.1007/7651_2015_313

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References

  1. Gardner RL (1985) Clonal analysis of early mammalian development. Philos Trans R Soc Lond B Biol Sci 312:163–178

    Article  CAS  PubMed  Google Scholar 

  2. Rossant J, Chazaud C, Yamanaka Y (2003) Lineage allocation and asymmetries in the early mouse embryo. Philos Trans R Soc Lond B Biol Sci 358:1341–1349

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Yamanaka Y, Ralston A, Stephenson RO, Rossant J (2006) Cell and molecular regulation of the mouse blastocyst. Dev Dyn 235:2301–2314

    Article  CAS  PubMed  Google Scholar 

  4. Gardner RL (1982) Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo. J Embryol Exp Morphol 68:175–198

    CAS  PubMed  Google Scholar 

  5. Gardner RL (1984) An in situ cell marker for clonal analysis of development of the extraembryonic endoderm in the mouse. J Embryol Exp Morphol 80:251–288

    CAS  PubMed  Google Scholar 

  6. Gardner RL, Rossant J (1979) Investigation of the fate of 4–5 day post-coitum mouse inner cell mass cells by blastocyst injection. J Embryol Exp Morphol 52:141–152

    CAS  PubMed  Google Scholar 

  7. Kwon GS, Viotti M, Hadjantonakis A-K (2008) The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages. Dev Cell 15:509–520

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Plusa B, Piliszek A, Frankenberg S, Artus J, Hadjantonakis A-K (2008) Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst. Development 135:3081–3091

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Chazaud C, Yamanaka Y, Pawson T, Rossant J (2006) Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev Cell 10:615–624

    Article  CAS  PubMed  Google Scholar 

  10. Kurimoto K, Yabuta Y, Ohinata Y, Ono Y, Uno KD, Yamada RG et al (2006) An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis. Nucleic Acids Res 34:e42

    Article  PubMed Central  PubMed  Google Scholar 

  11. Rossant J, Tam PPL (2009) Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development 136:701–713

    Article  CAS  PubMed  Google Scholar 

  12. Grabarek JB, Żyżyńska K, Saiz N, Piliszek A, Frankenberg S, Nichols J et al (2012) Differential plasticity of epiblast and primitive endoderm precursors within the ICM of the early mouse embryo. Development 139:129–139

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Chambers I, Silva J, Colby D, Nichols J, Nijmeijer B, Robertson M et al (2007) Nanog safeguards pluripotency and mediates germline development. Nature 450:1230–1234

    Article  CAS  PubMed  Google Scholar 

  14. Filipczyk A, Gkatzis K, Fu J, Hoppe PS, Lickert H, Anastassiadis K et al (2013) Biallelic expression of Nanog protein in mouse embryonic stem cells. Cell Stem Cell 13:12–13

    Article  CAS  PubMed  Google Scholar 

  15. Singh AM, Hamazaki T, Hankowski KE, Terada N (2007) A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem Cells 25:2534–2542

    Article  CAS  PubMed  Google Scholar 

  16. Canham MA, Sharov AA, Ko MSH, Brickman JM (2010) Functional heterogeneity of embryonic stem cells revealed through translational amplification of an early endodermal transcript. PLoS Biol 8:e1000379

    Article  PubMed Central  PubMed  Google Scholar 

  17. Martinez Arias A, Brickman JM (2011) Gene expression heterogeneities in embryonic stem cell populations: origin and function. Curr Opin Cell Biol 23:650–656

    Article  CAS  PubMed  Google Scholar 

  18. Morgani SM, Canham MA, Nichols J, Sharov AA, Migueles RP, Ko MSH et al (2013) Totipotent embryonic stem cells arise in ground-state culture conditions. Cell Rep 3:1945–1957

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Torres-Padilla M-E, Chambers I (2014) Transcription factor heterogeneity in pluripotent stem cells: a stochastic advantage. Development 141:2173–2181

    Article  CAS  PubMed  Google Scholar 

  20. Toyooka Y, Shimosato D, Murakami K, Takahashi K, Niwa H (2008) Identification and characterization of subpopulations in undifferentiated ES cell culture. Development 135:909–918

    Article  CAS  PubMed  Google Scholar 

  21. Berg DLC, van den Zhang W, Yates A, Engelen E, Takacs K, Bezstarosti K et al (2008) Estrogen-related receptor beta interacts with Oct4 to positively regulate Nanog gene expression. Mol Cell Biol 28:5986–5995

    Article  PubMed Central  PubMed  Google Scholar 

  22. Hayashi K, Lopes SM, Tang F, Surani MA (2008) Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3:391–401

    Article  CAS  PubMed  Google Scholar 

  23. Niwa H, Ogawa K, Shimosato D, Adachi K (2009) A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460:118–122

    Article  CAS  PubMed  Google Scholar 

  24. Zalzman M, Falco G, Sharova LV, Nishiyama A, Thomas M, Lee S-L et al (2010) Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464:858–863

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D et al (2012) ES cell potency fluctuates with endogenous retrovirus activity. Nature 487:57–63

    PubMed Central  CAS  PubMed  Google Scholar 

  26. Hooper M, Hardy K, Handyside A, Hunter S, Monk M (1987) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 326:292–295

    Article  CAS  PubMed  Google Scholar 

  27. Davies D (2007) Cell sorting by flow cytometry. In: Macey MG (ed) Flow cytometry. Humana Press, Totowa, NJ, pp 257–276

    Chapter  Google Scholar 

  28. Hoffman RA (2001) Pulse width for particle sizing. In Current protocols in cytometry. John Wiley & Sons, Inc., New York, pp. 1.23.1–1.23.17

    Google Scholar 

  29. Allen P, Davies D (2007) Apoptosis detection by flow cytometry. In: Macey MG (ed) Flow cytometry. Humana Press, Totowa, NJ, pp 147–163

    Chapter  Google Scholar 

  30. Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F (1997) Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1–20

    Article  CAS  PubMed  Google Scholar 

  31. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Majno G, Joris I (1995) Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 146:3–15

    PubMed Central  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. The Danish Stem Cell Centre—DanStem, University of Copenhagen, 3B Blegdamsvej, DK-2200, Copenhagen, Denmark

    Jurriaan Hölzenspies, Gelo Dela Cruz & Joshua M. Brickman

Authors
  1. Jurriaan Hölzenspies
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  2. Gelo Dela Cruz
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  3. Joshua M. Brickman
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Corresponding author

Correspondence to Joshua M. Brickman .

Editor information

Editors and Affiliations

  1. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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Hölzenspies, J., Cruz, G.D., Brickman, J.M. (2015). Resolving Heterogeneity: Fluorescence-Activated Cell Sorting of Dynamic Cell Populations from Feeder-Free Mouse Embryonic Stem Cell Culture. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 1341. Humana Press, New York, NY. https://doi.org/10.1007/7651_2015_254

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  • DOI: https://doi.org/10.1007/7651_2015_254

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2953-5

  • Online ISBN: 978-1-4939-2954-2

  • eBook Packages: Springer Protocols

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