Germ Cell Protocols pp 79-98

Part of the Methods in Molecular Biology™ book series (MIMB, volume 254)

Differentiating Embryonic Stem Cells into Embryoid Bodies

  • Gisele Höpfl
  • Max Gassmann
  • Isabelle Desbaillets

Abstract

Since the early 1980s, embryonic stem (ES) cells have been isolated from the inner cell mass of the preimplantation blastocyst (1, 2, 3). They can be maintained in a pluripotent state for indefinite periods of time in the presence of the leukemia inhibitory factor (LIF) or in coculture with mouse embryonic fibroblasts (MEFs). When cultured in the absence of LIF, ES cells differentiate spontaneously, forming three-dimensional (3D) aggregates called embryoid bodies (EBs, Fig. 1). EBs recapitulate many aspects of cell differentiation during early mammalian embryogenesis (4,5) and give rise to mature cells of all three germ layers (reviewed in ref.6). The EB system is an extremely valuable tool for the investigation of embryonic development in vitro because it is challenging and time-consuming to isolate early-stage cells from developing embryos. Moreover, EBs can be used when difficulties arise to determine the cellular basis for embryonic defects or the interpretation of knockout phenotypes, especially those complicated by early embryonic lethality. In addition, great efforts are made to define protocols to preferentially direct ES cell differentiation as EBs toward one single cell fate, therefore providing a source of normal cells and raising opportunities for therapeutic interventions to correct cell/tissue damage and/or dysfunction.

References

  1. 1.
    Evans M. J. and Kaufman M. H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156.PubMedCrossRefGoogle Scholar
  2. 2.
    Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638.PubMedCrossRefGoogle Scholar
  3. 3.
    Brook, F. A. and Gardner R. L. (1997) The origin and efficient derivation of embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA 94, 5709–5712.PubMedCrossRefGoogle Scholar
  4. 4.
    Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., and Kenter, 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.PubMedGoogle Scholar
  5. 5.
    Shen, M. M. and Leder, P. (1992) Leukemia inhibitory factor is expressed by the preimplantation uterus and selectively blocks primitive ectoderm formation in vitro. Proc. Natl. Acad. Sci. USA 89, 8240–8244.PubMedCrossRefGoogle Scholar
  6. 6.
    Desbaillets, I., Ziegler, U., Groscurth, P., and Gassmann, M. (2000) Embryoid bodies: an in vitro model of mouse embryogenesis. Exp. Physiol. 85, 645–651.PubMedCrossRefGoogle Scholar
  7. 7.
    Wiles, M. V. (1993) Embryonic stem cell differentiation in vitro. Methods Enzymol. 225, 900–918.PubMedCrossRefGoogle Scholar
  8. 8.
    Wong, P. M., Chung, S. W., Reicheld, S. M., and Chui, D. H. (1986) Hemoglobin switching during murine embryonic development: evidence for two populations of embryonic erythropoietic progenitor cells. Blood 67, 716–21.PubMedGoogle Scholar
  9. 9.
    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.PubMedGoogle Scholar
  10. 10.
    Wiles, M. V. and Keller, G. (1991) Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development 111, 259–267.PubMedGoogle Scholar
  11. 11.
    Weiss, M. J., Keller, G.. and Orkin, S. H. (1994) Novel insights into erythroid development revealed through in vitro differentiation of GATA-1 embryonic stem cells. Gene Dev. 8, 1184–1197.PubMedCrossRefGoogle Scholar
  12. 12.
    Potocnik, A. J., Nielsen, P. J., and Eichmann, K. (1994) In vitro generation of lymphoid precursors from embryonic stem cells. EMBO J. 13, 5274–5283.PubMedGoogle Scholar
  13. 13.
    Potocnik, A. J., Kohler, H., and Eichmann, K. (1997) Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells. Proc. Natl. Acad. Sci. USA 94, 10,295–10,300.PubMedCrossRefGoogle Scholar
  14. 14.
    Vittet, D., Prandini, M. H., Berthier, R., Schweitzer, A., Martin-Sistero H., Uzan, G., and Dejana, E. (1996) Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88, 3424–3431.PubMedGoogle Scholar
  15. 15.
    Gualandris, A., Annes, J. P., Arese, M., Noguera, I., Jurukovski V., and D. B. Rifkin (2000) The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium. Mol. Biol. Cell. 11, 4295–4308.PubMedGoogle Scholar
  16. 16.
    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.PubMedGoogle Scholar
  17. 17.
    Hescheler, J., Fleischmann, B. K., Lentini, S., Maltsev, V. A., Rohwedel, J., Wobus, A. M., and Addicks, K. (1997) Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc. Res. 36, 149–162.PubMedCrossRefGoogle Scholar
  18. 18.
    Wobus, A. M., Rohwedel, J., Maltsev, V., and Hescheler, J. (1994) In vitro differentiation of embryonic stem cells into cardiomyocytes or skeletal muscle is specifically modulated by retinoic acid. Roux’s Arch. Dev. Biol. 36–45.Google Scholar
  19. 19.
    Dani, C., Smith, A. G., Dessolin, S., Leroy, P., Staccini, L., Villageois, P., et al. (1997) Differentiation of embryonic stem cells into adipocytes in vitro. J. Cell Sci. 110, 1279–1285.PubMedGoogle Scholar
  20. 20.
    Rohwedel, J., Maltsev, V., Bober, E., Arnold, H. H., Hescheler J., and Wobus, A. M. (1994) Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev. Biol. 164, 87–101.PubMedCrossRefGoogle Scholar
  21. 21.
    Drab, M., Haller, H., Bychkov, R., Erdmann, B., Lindschau, C., Haase, H., et al. (1997) From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model. FASEB J. 11, 905–915.PubMedGoogle Scholar
  22. 22.
    Strubing, C., Ahnert-Hilger, G., Shan, J., Wiedenmann, B., Hescheler J., and Wobus, A. M. (1995) Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech. Dev. 53, 275–287.PubMedCrossRefGoogle Scholar
  23. 23.
    Strubing, C., Rohwedel, J., Ahnert-Hilger, G., Wiedenmann, B., Hescheler, J., and Wobus, A. M. (1997) Development of G protein-mediated Ca2+ channel regulation in mouse embryonic stem cell-derived neurons. Eur. J. Neurosci. 9, 824–832.PubMedCrossRefGoogle Scholar
  24. 24.
    Angelov, D. N., Arnhold, S., Andressen, C., Grabsch, H., Puschmann, M., Hescheler, J., and Addicks, K. (1998) Temporospatial relationships between macroglia and microglia during in vitro differentiation of murine stem cells. Dev. Neurosci. 20, 42–51.PubMedCrossRefGoogle Scholar
  25. 25.
    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.PubMedGoogle Scholar
  26. 26.
    Bain, G., Kitchens, D., Yao, M., Huettner, J. E., and Gottlieb, D. I. (1995) Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342–357.PubMedCrossRefGoogle Scholar
  27. 27.
    Bain, G., Ray, W. J., Yao, M., and Gottlieb, D. I. (1996) Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem. Biophy.s Res. Commun. 223, 691–694.CrossRefGoogle Scholar
  28. 28.
    Brustle, O., Spiro, A. C., Karram, K., Choudhary, K., Okabe, S., and McKay, R. D. (1997) In vitro-generated neural precursors participate in mammalian brain development. Proc. Natl. Acad. Sci. USA 94, 14,809–14,814.PubMedCrossRefGoogle Scholar
  29. 29.
    Rolletschek, A., Chang, H., Guan, K., Czyz, J., Meyer, M., and Wobus, A. M. (2001) Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors. Mech. Dev. 105, 93–104.PubMedCrossRefGoogle Scholar
  30. 30.
    Gajovic, S., St-Onge, L., Yokota, Y.,and Gruss, P. (1997) Retinoic acid mediates Pax6 expression during in vitro differentiation of embryonic stem cells. Differentiation 62, 187–192.PubMedGoogle Scholar
  31. 31.
    Okabe, S., Forsberg-Nilsson, K., Spiro, A.C., Segal, M., and McKay, R. D. (1996) Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech. Dev. 59, 89–102.PubMedCrossRefGoogle Scholar
  32. 32.
    Lee, S. H., Lumelsky, N., Studer, L., Auerbach, J. M., and McKay, R. D. (2000) Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat. Biotechnol. 18, 675–679.PubMedCrossRefGoogle Scholar
  33. 33.
    Phillips, B. W., Belmonte, N., Vernochet, C., Ailhaud, G., and Dani, C. (2001) Compactin enhances osteogenesis in murine embryonic stem cells. Biochem. Biophys. Res. Commun. 284, 478–484.PubMedCrossRefGoogle Scholar
  34. 34.
    Kramer, J., Hegert, C., Guan, K., Wobus, A. M., Muller, P. K., and Rohwedel, J. (2000) Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech. Dev. 92, 193–205.PubMedCrossRefGoogle Scholar
  35. 35.
    Tsai, M., Wedemeyer, J., Ganiatsas, S., Tam, S. Y., Zon, L. I., and Galli, S. J. (2000) In vivo immunological function of mast cells derived from embryonic stem cells: an approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo. Proc. Natl. Acad. Sci. USA 97, 9186–9190.PubMedCrossRefGoogle Scholar
  36. 36.
    Fairchild, P. J., Brook, F. A., Gardner, R. L., Graca, L., Strong, V., Tone, Y., et al. (2000) Directed differentiation of dendritic cells from mouse embryonic stem cells. Curr. Biol. 10, 1515–1518.PubMedCrossRefGoogle Scholar
  37. 37.
    Hamazaki, T., Iiboshi, Y., Oka, M., Papst, P. J., Meacham, A. M., Zon, L. I., and Terada, N. (2001) Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS Lett. 497, 15–19.PubMedCrossRefGoogle Scholar
  38. 38.
    Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R.,and McKay, R. (2001) Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292, 1389–1394.PubMedCrossRefGoogle Scholar
  39. 39.
    Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N., and Keller, G. (1997) A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 386, 488–493.PubMedCrossRefGoogle Scholar
  40. 40.
    Tsai F. Y. and Orkin S. H. (1997) Transcription factor GATA-2 is required for proliferation/survival of early hematopoietic cells and mast cell formation, but not for erythroid and myeloid terminal differentiation. Blood 89, 3636–3643.PubMedGoogle Scholar
  41. 41.
    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.PubMedCrossRefGoogle Scholar
  42. 42.
    Gassmann, M., Fandrey, J., Bichet, S., Wartenberg, M.,. Marti, H. H, Bauer, C., et al. (1996) Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells. Proc. Natl. Acad. Sci. USA 93, 2867–2872.PubMedCrossRefGoogle Scholar
  43. 43.
    Brustle, O., Jones, K. N., Learish, R. D., Karram, K., Choudhary, K., Wiestler, O. D., et al. (1999) Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science 285, 754–756.PubMedCrossRefGoogle Scholar
  44. 44.
    Rohwedel, J., Guan, K., and Wobus, A. M. (1999) Induction of cellular differentiation by retinoic acid in vitro. Cells Tissues Organs 165, 190–202.PubMedCrossRefGoogle Scholar
  45. 45.
    McDonald, J. W., Liu, X. Z., Qu, Y., Liu, S., Mickey, S. K., Turetsky, D., et al. (1999) Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med. 5, 1410–1412.PubMedCrossRefGoogle Scholar
  46. 46.
    S. Liu, Y. Qu, T. J. Stewart, M. J. Howard, S. Chakrabortty, T. F. Holekamp and J. W. McDonald (2000) Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc. Natl. Acad. Sci. USA 97, 6126–6131.PubMedCrossRefGoogle Scholar
  47. 47.
    van Inzen, W. G., Peppelenbosch, M. P., van den Brand, M. W., Tertoolen, L. G., and de Laat, S. W. (1996) Neuronal differentiation of embryonic stem cells. Biochim. Biophys. Acta. 1312, 21–26.PubMedCrossRefGoogle Scholar
  48. 48.
    Johansson, B. M. and Wiles, M. V. (1995) Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development. Mol. Cell Biol. 15, 141–151.PubMedGoogle Scholar
  49. 49.
    Adelman, C. A., Chattopadhyay, S., and Bieker, J. J. (2002) The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media. Development 129, 539–549.PubMedGoogle Scholar
  50. 50.
    Bichet, S., Wenger, R. H., Camenisch, G., Rolfs, A., Ehleben, W., Porwol T., et al. (1999) Oxygen tension modulates beta-globin switching in embryoid bodies. FASEB J. 13, 285–295.PubMedGoogle Scholar
  51. 51.
    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.PubMedGoogle Scholar
  52. 52.
    Fujiwara, Y., Browne, C. P., Cunniff, K., Goff, S. C., and Orkin S. H. (1996) Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc. Natl. Acad. Sci. USA 93, 12,355–12,358.PubMedCrossRefGoogle Scholar
  53. 53.
    Zhang, S. C., Wernig, M., Duncan, I. D., Brustle O., and Thomson, J. A. (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Gisele Höpfl
    • 1
  • Max Gassmann
    • 2
  • Isabelle Desbaillets
    • 2
  1. 1.Institute of PhysiologyUniversity of ZürichZürichSwitzerland
  2. 2.Institute of Veterinary PhysiologyUniversity of ZürichZürichSwitzerland

Personalised recommendations