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The Culture of Mouse Embryonic Stem Cells and Formation of Embryoid Bodies

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Mouse Cell Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 633))

Abstract

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of the pre-implantation blastocyst. They have the capacity to undergo indefinite rounds of self-renewing cell division and differentiate into all the cell lineages of the developing embryo. In suspension culture, ES cells will differentiate into aggregates known as embryoid bodies in a manner similar to the early embryo. This culture system therefore provides a useful model to study the relatively inaccessible stages of mammalian development. We describe methods for the routine maintenance of mouse embryonic stem cells in culture, assays of stem cell self-renewal potential in monolayer culture and the generation of embryoid bodies to study differentiation pathways.

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References

  1. Thomas, K. R., and Capecchi, M. R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512.

    Article  CAS  PubMed  Google Scholar 

  2. Capecchi, M. R. (1989) The new mouse genetics: altering the genome by gene targeting. Trends Genet. 5, 70–76.

    Article  CAS  PubMed  Google Scholar 

  3. Misra, R. P., and Duncan, S. A. (2002) Gene targeting in the mouse: advances in introduction of transgenes into the genome by homologous recombination. Endocrine 19, 229–238.

    Article  CAS  PubMed  Google Scholar 

  4. Kwan, K. M. (2002) Conditional alleles in mice: practical considerations for tissue-specific knockouts. Genesis 32, 49–62.

    Article  CAS  PubMed  Google Scholar 

  5. Evans, M. J., and Kaufman, M. H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156.

    Article  CAS  PubMed  Google Scholar 

  6. Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638.

    Article  CAS  PubMed  Google Scholar 

  7. Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A., and Gough, N. M. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687.

    Article  CAS  PubMed  Google Scholar 

  8. Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G., Moreau, J., Stahl, M., and Rogers, D. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688–690.

    Article  CAS  PubMed  Google Scholar 

  9. Nichols, J., Evans, E. P., Smith, A. G. (1990) Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 110, 1341–1348.

    CAS  PubMed  Google Scholar 

  10. 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  PubMed  Google Scholar 

  11. Niwa, H., Burdon, T., Chambers, I., and Smith, A. (1998) Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 12, 2048–2060.

    Article  CAS  PubMed  Google Scholar 

  12. Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., and Kodama, H. (1998) Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 125, 1747–1757.

    CAS  PubMed  Google Scholar 

  13. Fok, E. Y., and Zandstra, P. W. (2005) Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation. Stem Cells 23, 1333–1342.

    Article  CAS  PubMed  Google Scholar 

  14. Chambers, I., Silva, J., Colby, D., Nichols, J., Nijmeijer, B., Robertson, M., Vrana, J., Jones, K., Grotewold, L., and Smith, A. (2007) Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234.

    Article  CAS  PubMed  Google Scholar 

  15. Wobus, A. M., and Boheler, K. R. (2005) Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol. Rev. 85, 635–678.

    Article  CAS  PubMed  Google Scholar 

  16. Smith, A. G. (2001) Embryo-derived stem cells: of mice and men. Ann. Rev. Cell Dev. Biol. 17, 435–462.

    Article  CAS  Google Scholar 

  17. Lerou, P. H., and Daley, G. Q. (2005) Therapeutic potential of embryonic stem cells. Blood Rev. 19, 321–331.

    Article  PubMed  Google Scholar 

  18. Liew, C. G., Moore, H., Ruban, L., Shah, N., Cosgrove, K., Dunne, M., and Andrews, P. (2005) Human embryonic stem cells: possibilities for human cell transplantation. Ann. Med. 37, 521–532.

    Article  CAS  PubMed  Google Scholar 

  19. Mountford, P., Zevnik, B., Duwel, A., Nichols, J., Li, M., Dani, C., Robertson, M., Chambers, I., and Smith, A. (1994) Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc. Natl. Acad. Sci. USA 91, 4303–4307.

    Article  CAS  PubMed  Google Scholar 

  20. Jones, E. A., Tosh, D., Wilson, D. I., Lindsay, S., and Forrester, L. M. (2002) Hepatic differentiation of murine embryonic stem cells. Exp. Cell Res. 272, 15–22.

    Article  CAS  PubMed  Google Scholar 

  21. Fehling, H. J., Lacaud, G., Kubo, A., Kennedy, M., Robertson, S., Keller, G., and Kouskoff, V. (2003) Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217–4227.

    Article  CAS  PubMed  Google Scholar 

  22. Tada, S., Era, T., Furusawa, C., Sakurai, H., Nishikawa, S., Kinoshita, M., Nakao, K., and Chiba, T. (2005) Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132, 4363–4374.

    Article  CAS  PubMed  Google Scholar 

  23. Rohwedel, J., Guan, K., Zuschratter, W., Jin, S., Ahnert-Hilger, G., Furst, D., Fassler, R., and Wobus, A. M. (1998) Loss of beta1 integrin function results in a retardation of myogenic, but an acceleration of neuronal, differentiation of embryonic stem cells in vitro. Dev. Biol. 201, 167–184.

    Article  CAS  PubMed  Google Scholar 

  24. Jackson, M., Baird, J. W., Cambray, N., Ansell, J. D., Forrester, L. M., and Graham, G. J. (2002) Cloning and characterization of Ehox, a novel homeobox gene essential for embryonic stem cell differentiation. J. Biol. Chem. 277, 38683–38692.

    Article  CAS  PubMed  Google Scholar 

  25. Lacaud, G., Gore, L., Kennedy, M., Kouskoff, V., Kingsley, P., Hogan, C., Carlsson, L., Speck, N., Palis, J., and Keller, G. (2002) Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood 100, 458–466.

    Article  CAS  PubMed  Google Scholar 

  26. Jackson, M., Krassowska, A., Gilbert, N., Chevassut, T., Forrester, L., Ansell, J., and Ramsahoye, B. (2004) Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells. Mol. Cell Biol. 24, 8862–8871.

    Article  CAS  PubMed  Google Scholar 

  27. Capo-Chichi, C. D., Rula, M. E., Smedberg, J. L., Vanderveer, L., Parmacek, M. S., Morrisey, E. E., Godwin, A. K., Xu, X. X. (2005) Perception of differentiation cues by GATA factors in primitive endoderm lineage determination of mouse embryonic stem cells. Dev. Biol. 286, 574–586.

    Article  CAS  PubMed  Google Scholar 

  28. Brennan, J., and Skarnes, W. C. (2008) Gene trapping in mouse embryonic stem cells. Methods Mol. Biol. 461, 133–148.

    Article  CAS  PubMed  Google Scholar 

  29. Hadjantonakis, A. K., Pirity, M., and Nagy, A. (2008) Cre recombinase mediated alterations of the mouse genome using embryonic stem cells. Methods Mol. Biol. 461, 111–132.

    Article  CAS  PubMed  Google Scholar 

  30. Wobus, A. M., Kaomei, G., Shan, J., Wellner, M. C., Rohwedel, J., Ji, G., Fleischmann, B., Katus, H. A., Hescheler, J., and Franz, W. M. (1997) Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J. Mol. Cell. Cardiol. 29, 1525–1539.

    Article  CAS  PubMed  Google Scholar 

  31. Keller, G., Kennedy, M., Papayannopoulou, T., and Wiles, M. V. (1993) Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol. Cell. Biol. 13, 473–486.

    CAS  PubMed  Google Scholar 

  32. Kitajima, K., Tanaka, M., Zheng, J., Sakai-Ogawa, E., and Nakano, T. (2003) In vitro differentiation of mouse embryonic stem cells to hematopoietic cells on an OP9 stromal cell monolayer. Methods Enzymol. 365, 72–83.

    Article  PubMed  Google Scholar 

  33. Honda, M., Hamazaki, T. S., Komazaki, S., Kagechika, H., Shudo, K., and Asashima, M. (2005) RXR agonist enhances the differentiation of cardiomyocytes derived from embryonic stem cells in serum-free conditions. Biochem. Biophys. Res. Commun. 333, 1334–1340.

    Article  CAS  PubMed  Google Scholar 

  34. Yasunaga, M., Tada, S., Torikai-Nishikawa, S., Nakano, Y., Okada, M., Jakt, L. M., Nishikawa, S., Chiba, T., Era, T., and Nishikawa, S. (2005) Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat. Biotechnol. 23, 1542–1550.

    Article  CAS  PubMed  Google Scholar 

  35. Gouon-Evans, V., Boussemart, L., Gadue, P., Nierhoff, D., Koehler, C. I., Kubo, A., Shafritz, D. A., and Keller, G. (2006) BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat. Biotechnol. 24, 1402–1411.

    Article  CAS  PubMed  Google Scholar 

  36. Andäng, M., Moliner, A., Doege, C. A., Ibañez, C. F., and Ernfors, P. (2008) Optimized mouse ES cell culture system by suspension growth in a fully defined medium. Nat. Protoc. 3, 1013–1017.

    Article  PubMed  Google Scholar 

  37. Hooper, M. L. (1992) Embryonal carcinoma and Embryonal stem cells. In “Embryonal stem cells: introducing planned changes into the animal germline,” Evans, H. J. (Ed.). Vol. 1, Harwood Academic Publishers, Switzerland.

    Google Scholar 

  38. Martin, G. R., and Evans, M. J. (1975) Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc. Natl. Acad. Sci. USA 72, 1441–14415.

    Article  CAS  PubMed  Google Scholar 

  39. Wallukat, G., and Wobus, A. (1991) Use of spontaneously beating heart muscle cells differentiating from pluripotential embryonic stem cells for testing of chronotropic agents. Arch. Toxicol. Suppl. 14, 136–139.

    CAS  PubMed  Google Scholar 

  40. Maltsev, V. A., Wobus, A. M., Rohwedel, J., Bader, M., and Hescheler, J. (1994) Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ. Res. 75, 233–244.

    CAS  PubMed  Google Scholar 

  41. Vittet, D., Prandini, M. H., Berthier, R., Schweitzer, A., Martin-Sisteron, H., Uzan, G., and Dejana, E. (1996) Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88, 3424–3431.

    CAS  PubMed  Google Scholar 

  42. Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87, 27–45.

    CAS  PubMed  Google Scholar 

  43. Wiles, M. V., andKeller, G. (1991) Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development 111, 259–267.

    CAS  PubMed  Google Scholar 

  44. Field, S. J., Johnson, R. S., Mortensen, R. M., Papaioannou, V. E., Spiegelman, B. M., and Greenberg, M. E. (1992) Growth and differentiation of embryonic stem cells that lack an intact c-fos gene. Proc. Natl. Acad. Sci. USA 89, 9306–9310.

    Article  CAS  PubMed  Google Scholar 

  45. Dani, C., Smith, A. G., Dessolin, S., Leroy, P., Staccini, L., Villageois, P., Darimont, C., and Ailhaud, G. (1997) Differentiation of embryonic stem cells into adipocytes in vitro. J. Cell Sci. 110, 1279–1285.

    CAS  PubMed  Google Scholar 

  46. Bain, G., Kitchens, D., Yao, M., Huettner, J. E., Gottlieb, D. I. (1995) Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342–357.

    Article  CAS  PubMed  Google Scholar 

  47. Fraichard, A., Chassande, O., Bilbaut, G., Dehay, C., Savatier, P., and Samarut, J. (1995) In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 108, 3181–3188.

    CAS  PubMed  Google Scholar 

  48. Kubo, A., Shinozaki, K., Shannon, J. M., Kouskoff, V., Kennedy, M., Woo, S., Fehling, H. J., and Keller, G. (2004) Development of definitive endoderm from embryonic stem cells in culture. Development 131, 1651–1562.

    Article  CAS  PubMed  Google Scholar 

  49. Blyszczuk, P., Asbrand, C., Rozzo, A., Kania, G., St-Onge, L., Rupnik, M., and Wobus, A. M. (2004) Embryonic stem cells differentiate into insulin-producing cells without selection of nestin-expressing cells. Int. J. Dev. Biol. 48, 1095–1104.

    Article  CAS  PubMed  Google Scholar 

  50. Dang, S. M., Kyba, M., Perlingeiro, R., Daley, G. Q., and Zandstra, P. W. (2002) Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems. Biotechnol. Bioeng. 78, 442–453.

    Article  CAS  PubMed  Google Scholar 

  51. Boheler, K. R., Crider, D. G., Tarasova, Y., and Maltsev, V. A. (2005) Cardiomyocytes derived from embryonic stem cells. Methods Mol. Med. 108, 417–435.

    PubMed  Google Scholar 

  52. Nakano, T., Kodama, H., and Honjo, T. (1994) Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101.

    Article  CAS  PubMed  Google Scholar 

  53. Kawase, E., Suemori, H., Takahashi, N., Okazaki, K., Hashimoto, K., and Nakatsuji, N. (1994) Strain difference in establishment of mouse embryonic stem (ES) cell lines. Int. J. Dev. Biol. 38, 385–390.

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  55. Handyside, A. H., O’Neil, G. T., Jones, M., and Hooper, M. L. (1989) Use of BRL-conditioned medium in combination with feeder layers to isolate a diploid embryonal stem cell line. Rouxs Arch. Dev. Biol. 198, 48–56.

    Article  Google Scholar 

  56. Robertson, E., and Bradley, A. (1986) Production of permanent cell lines from early embryos and their use in studying developmental problems. In “Experimental approaches to mammalian embryonic development,” Rossant, J., and Pederson, R. A. (Eds.). Cambridge University Press, Cambridge, UK, pp. 475–506.

    Google Scholar 

  57. Joyner, A. L. (2000) Gene targeting: a practical approach. Oxford University Press, Oxford.

    Google Scholar 

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Author information

Authors and Affiliations

  1. John Hughes Bennett Laboratory, Queen’s Medical Research Institute, University of Edinburgh, EH164TJ, Edinburgh, UK

    Melany Jackson, A. Helen Taylor & Lesley M. Forrester

  2. Department of Medical Genetics and Regional Genetic Service, Central Manchester NHS Foundation Trust, St Mary’s Hospital, Manchester, M13 0JH, UK

    Elizabeth A. Jones

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  1. Melany Jackson
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  2. A. Helen Taylor
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  3. Elizabeth A. Jones
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  4. Lesley M. Forrester
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Editors and Affiliations

  1. Dept. Biology & Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom

    Andrew Ward

  2. Dept. Biology & Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom

    David Tosh

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Jackson, M., Taylor, A.H., Jones, E.A., Forrester, L.M. (2010). The Culture of Mouse Embryonic Stem Cells and Formation of Embryoid Bodies. In: Ward, A., Tosh, D. (eds) Mouse Cell Culture. Methods in Molecular Biology, vol 633. Humana Press. https://doi.org/10.1007/978-1-59745-019-5_1

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  • DOI: https://doi.org/10.1007/978-1-59745-019-5_1

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  • Print ISBN: 978-1-58829-772-3

  • Online ISBN: 978-1-59745-019-5

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