Skip to main content
SpringerLink
Log in

Propagation and Maintenance of Mouse Embryonic Stem Cells

  • Protocol
  • First Online:
Mouse Cell Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1940))

Abstract

Mouse embryonic stem cells (mESCs) are pluripotent cells derived from preimplantation embryos that have the capacity to self-renew indefinitely in vitro. mESCs are an indispensable tool for studying cellular differentiation in vitro, generating disease in a dish models, and have been used extensively for the generation of transgenic animals. Therefore, maintaining their pluripotent state, even after extended culture, is crucial for their utility. Herein, we describe in detail a protocol for the culture of mESCs in the presence of fetal calf serum (FCS), leukemia inhibitory factor (LIF), and a layer of irradiated mouse embryonic fibroblasts (iMEFs). This culture system reliably sustains mESC pluripotency and self-renewal capacity, allowing their use in a wide range of experimental settings.

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

Access this chapter

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 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
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

Institutional subscriptions

References

  1. Hackett JA, Surani MA (2014) Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 15:416–430

    Article  CAS  Google Scholar 

  2. Evans M (2011) Discovering pluripotency: 30 years of mouse embryonic stem cells. Nat Rev Mol Cell Biol 12:680–686

    Article  CAS  Google Scholar 

  3. Stevens LC, Little CC (1954) Spontaneous testicular teratomas in an inbred strain of mice. Proc Natl Acad Sci U S A 40:1080–1087

    Article  CAS  Google Scholar 

  4. Kleinsmith LJ, Pierce GB (1964) Multipotentiality of single embryonal carcinoma cells. Cancer Res 24:1544–1551

    CAS  PubMed  Google Scholar 

  5. Rosenthal MD, Wishnow RM, Sato GH (1970) In vitro growth and differentiation of clonal populations of multipotential mouse cells derived from a transplantable testicular teratocarcinoma. J Natl Cancer Inst 44:1001–1014

    CAS  PubMed  Google Scholar 

  6. Kahan BW, Ephrussi B (1970) Developmental potentialities of clonal in vitro cultures of mouse testicular teratoma. J Natl Cancer Inst 44:1015–1036

    CAS  PubMed  Google Scholar 

  7. Martin GR, Smith S, Epstein CJ (1978) Protein synthetic patterns in teratocarcinoma stem cells and mouse embryos at early stages of development. Dev Biol 66:8–16

    Article  CAS  Google Scholar 

  8. Martin GR, Evans MJ (1975) Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc Natl Acad Sci U S A 72(4):1441–1445

    Article  CAS  Google Scholar 

  9. Martin GR, Evans MJ (1975) Multiple differentiation of clonal teratocarcinoma stem cells following embryoid body formation in vitro. Cell 6:467–474

    Article  Google Scholar 

  10. Adamson ED, Evans MJ, Magrane GG (1977) Biochemical markers of the progress of differentiation in cloned teratocarcinoma cell lines. Eur J Biochem 79:607–615

    Article  CAS  Google Scholar 

  11. Jacob F (1977) Mouse teratocarcinoma and embryonic antigens. Immunol Rev 33:3–32

    Article  CAS  Google Scholar 

  12. Kapadia A, Feizi T, Evans MJ (1981) Changes in the expression and polarization of blood group I and i antigens in post-implantation embryos and teratocarcinomas of mouse associated with cell differentiation. Exp Cell Res 131:185–195

    Article  CAS  Google Scholar 

  13. Lovell-Badge R, Evans M (1980) Changes in protein synthesis during differentiation of embryonal carcinoma cells, and a comparison with embryo cells. Development 59:187–206

    CAS  Google Scholar 

  14. Stern PL, Willison KR, Lennox E, Galfrè G, Milstein C et al (1978) Monoclonal antibodies as probes for differentiation and tumor-associated antigens: a Forssman specificity on teratocarcinoma stem cells. Cell 14:775–783

    Article  CAS  Google Scholar 

  15. Papaioannou VE, McBurney MW, Gardner RL, Evans MJ (1975) Fate of teratocarcinoma cells injected into early mouse embryos. Nature 258:70–73

    Article  CAS  Google Scholar 

  16. Mintz B, Illmensee K (1975) Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc Natl Acad Sci U S A 72:3585–3589

    Article  CAS  Google Scholar 

  17. Brinster RL (1974) The effect of cells transferred into the mouse blastocyst on subsequent development. J Exp Med 140:1049

    Article  CAS  Google Scholar 

  18. Martin GR (1980) Teratocarcinomas and mammalian embryogenesis. Science 209:768–776

    Article  CAS  Google Scholar 

  19. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156

    Article  CAS  Google Scholar 

  20. Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78:7634–7638

    Article  CAS  Google Scholar 

  21. Bradley A, Evans M, Kaufman MH, Robertson E (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309:255–256

    Article  CAS  Google Scholar 

  22. Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A 90:8424–8428

    Article  CAS  Google Scholar 

  23. Horie K, Kokubu C, Yoshida J, Akagi K, Isotani A et al (2011) A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening. Nat Methods 8:1071–1077

    Article  CAS  Google Scholar 

  24. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918

    Article  CAS  Google Scholar 

  25. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L et al (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159:440–455

    Article  CAS  Google Scholar 

  26. Oji A, Noda T, Fujihara Y, Miyata H, Kim YJ et al (2016) CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice. Sci Rep 6:31666

    Article  CAS  Google Scholar 

  27. Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL et al (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336:684–687

    Article  CAS  Google Scholar 

  28. Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J et al (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688–690

    Article  CAS  Google Scholar 

  29. Rathjen PD, Toth S, Willis A, Heath JK, Smith AG (1990) Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage. Cell 62:1105–1114

    Article  CAS  Google Scholar 

  30. Martello G, Smith A (2014) The nature of embryonic stem cells. Annu Rev Cell Dev Biol 30:647–675

    Article  CAS  Google Scholar 

  31. 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  Google Scholar 

  32. Ye S, Li P, Tong C, Ying QL (2013) Embryonic stem cell self-renewal pathways converge on the transcription factor Tfcp2l1. EMBO J 32:2548–2560

    Article  CAS  Google Scholar 

  33. Martello G, Bertone P, Smith A (2013) Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor. EMBO J 32:2561–2574

    Article  CAS  Google Scholar 

  34. Tai C-I, Ying Q-L (2013) Gbx2, a LIF/Stat3 target, promotes reprogramming to and retention of the pluripotent ground state. J Cell Sci 126:1093–1098

    Article  CAS  Google Scholar 

  35. Ying Q-L, Nichols J, Chambers I, Smith A (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115:281–292

    Article  CAS  Google Scholar 

  36. Malaguti M, Nistor PA, Blin G, Pegg A, Zhou X, Lowell S (2013) Bone morphogenic protein signalling suppresses differentiation of pluripotent cells by maintaining expression of E-Cadherin. elife 2:e01197

    Article  Google Scholar 

  37. Ying Q-L, Wray J, Nichols J, Batlle-Morera L, Doble B et al (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–523

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Nefzger CM, Rossello FJ, Chen J, Liu X, Knaupp AS et al (2017) Cell Rep. 21:2649–2660.

    Google Scholar 

  40. Nefzger CM, Alaei S, Knaupp AS, Holmes ML, Polo JM (2014) Cell surface marker mediated purification of iPS cell intermediates from a reprogrammable mouse model. J Vis Exp (91):e51728.

    Google Scholar 

  41. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J (2003) Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 5:741–747

    Article  CAS  Google Scholar 

  42. Nefzger CM, Haynes JM, Pouton CW (2011) Directed expression of Gata2, Mash1, and Foxa2 synergize to induce the serotonergic neuron phenotype during in vitro differentiation of embryonic stem cells. Stem Cells 29:928–939

    Article  CAS  Google Scholar 

  43. Nefzger CM, Alaei S, Polo JM (2015) Isolation of reprogramming intermediates during generation of induced pluripotent stem cells from mouse embryonic fibroblasts. Methods Mol Biol 1330:205–218

    Article  CAS  Google Scholar 

  44. Alaei S, Knaupp A, Lim S, Chen J, Holmes M et al (2016) An improved reprogrammable mouse model harbouring the reverse tetracycline-controlled transcriptional transactivator 3. Stem Cell Res 17:49–53

    Article  CAS  Google Scholar 

  45. Firas J, Liu X, Nefzger CM, Polo JM (2014) GM-CSF and MEF-conditioned media support feeder-free reprogramming of mouse granulocytes to iPS cells. Differentiation 87:193–199

    Article  CAS  Google Scholar 

  46. Chen J, Nefzger CM, Rossello FJ, Sun YBY, Lim SM et al (2018) Fine tuning of canonical Wnt stimulation enhances differentiation of pluripotent stem cells independent of β-catenin-mediated T-Cell factor signaling. Stem Cells 36:822–833

    Article  Google Scholar 

Download references

Author information

Author notes

  1. Jacob M. Paynter and Joseph Chen contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia

    Jacob M. Paynter, Joseph Chen, Xiaodong Liu & Christian M. Nefzger

  2. Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia

    Jacob M. Paynter, Joseph Chen, Xiaodong Liu & Christian M. Nefzger

  3. Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia

    Jacob M. Paynter, Joseph Chen, Xiaodong Liu & Christian M. Nefzger

  4. Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia

    Christian M. Nefzger

Authors
  1. Jacob M. Paynter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaodong Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christian M. Nefzger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christian M. Nefzger .

Editor information

Editors and Affiliations

  1. Lung Health Research Centre, Department of Pharmacology and Therapeutics, University of Melbourne, Melbourne, VIC, Australia

    Ivan Bertoncello

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Paynter, J.M., Chen, J., Liu, X., Nefzger, C.M. (2019). Propagation and Maintenance of Mouse Embryonic Stem Cells. In: Bertoncello, I. (eds) Mouse Cell Culture. Methods in Molecular Biology, vol 1940. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9086-3_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9086-3_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9085-6

  • Online ISBN: 978-1-4939-9086-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation