Skip to main content
SpringerLink
Log in

Differentiation of Human Embryonic Stem Cells into Embryoid Bodies Comprising the Three Embryonic Germ Layers

  • Original Articles
  • Published:
Molecular Medicine Aims and scope Submit manuscript

Abstract

Background

Embryonic stem (ES) cells are lines of cells that are isolated from blastocysts. The murine ES cells were demonstrated to be true pluripotent cells as they differentiate into all embryonic lineages. Yet, in vitro differentiation of rhesus ES cells was somewhat inconsistent and disorganized. The recent isolation of human ES cells calls for exploring their pluripotential nature.

Materials and Methods

Human ES cells were grown in suspension to induce their differentiation into embryoid bodies (EBs). The differentiation status of the human ES cells and EBs was analyzed by following the expression pattern of several lineage-specific molecular markers using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization.

Results

Here we report the induction in vitro of cystic embryoid bodies from human ES cells. Our findings demonstrate induction of expression of cell-specific genes during differentiation of the human ES cells into EBs. In the human EBs, we could show a characteristic regional expression of embryonic markers specific to different cellular lineages, namely, ζ-globin (mesoderm), neurofilament 68Kd (ectoderm), and α-fetoprotein (endoderm). Moreover, we present a synchronously pulsing embryoid body that expresses the myocardium marker α-cardiac actin. In addition, dissociating the embryoid bodies and plating the cells as monolayers results in multiple morphologies, among them cells with neuronal appearance that express neurofilament 68Kd chain.

Conclusion

Human ES cells can reproducibly differentiate in vitro into EBs comprising the three embryonic germ layers. The ability to induce formation of human embryoid bodies that contain cells of neuronal, hematopoietic and cardiac origins will be useful in studying early human embryonic development as well as in transplantation medicine.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Robertson EJ. (1987) Embryo-derived stem cell lines. In: Robertson EJ (ed). Teratocarcinomas and Embryonic Stem Cells, a Practical Approach. IRL Press, Oxford, pp. 71–112.

    Google Scholar 

  2. Dushnik-Levinson M, Benvenisty N. (1995) Embryogenesis in vitro: study of differentiation of embryonic stem cells. Biol. Neonate 67: 77–83.

    Article  CAS  PubMed  Google Scholar 

  3. Capecchi MR. (1989) Altering the genome by homologous recombination. Science 244: 1288–1292.

    Article  CAS  PubMed  Google Scholar 

  4. Rossant J, Joyner AL. (1989) Towards a molecular-genetic analysis of mammalian development. Trends Genet. 5: 277–283.

    Article  CAS  PubMed  Google Scholar 

  5. Wobus AM, Holzhausen H, Jakel P, Schoneich J. (1984) Characterization of a pluripotent stem cell line derived from a mouse embryo. Exp. Cell. Res. 152: 212–219.

    Article  CAS  PubMed  Google Scholar 

  6. Wiles MV, Keller G. (1991) Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development 111: 259–267.

    PubMed  CAS  Google Scholar 

  7. Lindenbaum MH, Grosveld F. (1990) An in vitro globin gene switching model based on differentiated embryonic stem cells. Genes Dev. 4: 2075–2085.

    Article  CAS  PubMed  Google Scholar 

  8. Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. (1995) Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168: 342–357.

    Article  CAS  PubMed  Google Scholar 

  9. Levinson-Dushnik M, Benvenisty N. (1997) Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells. Mol. Cell. Biol. 17: 3817–3822.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147.

    Article  CAS  PubMed  Google Scholar 

  11. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Hearn JP. (1996) Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol. Reprod. 55: 254–259.

    Article  CAS  PubMed  Google Scholar 

  12. Thomson JA, Kalishman J, Golos TG, et al. (1995) Isolation of a primate embryonic stem cell line. Proc. Natl. Acad. Sci. U.S.A. 92: 7844–7848.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Thomson JA, Marshall VS. (1998) Primate embryonic stem cells. Curr. Top. Dev. Biol. 38: 133–165.

    Article  CAS  PubMed  Google Scholar 

  14. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299.

    Article  CAS  PubMed  Google Scholar 

  15. Southern EM. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503–517.

    Article  CAS  PubMed  Google Scholar 

  16. Grifman M, Galyam N, Seidman S, Soreq H. (1998) Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis. Proc. Natl. Acad. Sci. U.S.A. 95: 13935–13940.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Krumlauf R, Hammer RE, Tilghman SM, Brinster RL. (1985) Developmental regulation of α-fetoprotein genes in transgenic mice. Mol. Cell. Biol. 5: 1639–1648.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Leder A, Weir L, Leder P. (1985) Characterization, expression, and evolution of the mouse embryonic ζ-globin gene. Mol. Cell. Biol. 5: 1025–1033.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sassoon DA, Garner I, Buckingham M. (1988) Transcripts of alpha-cardiac and α-skeletal actins are early markers for myogenesis in the mouse embryo. Development 104: 155–164.

    PubMed  CAS  Google Scholar 

  20. Julien JP, Meyer D, Flavell D, Hurst J, Grosveld F. (1986) Cloning and developmental expression of the murine neurofilament gene family. Br. Res. 387: 243–250.

    CAS  Google Scholar 

  21. Shamblott MJ, Axelman J, Wang S, et al. (1998) Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA 95: 13726–13731.

    Article  CAS  PubMed  Google Scholar 

  22. Sanchez A, Jones WK, Gulick J, Doetschman T, Robbins J. (1991) Myosin heavy chain gene expression in mouse embryoid bodies. An in vitro developmental study. J. Biol. Chem. 266: 22419–22426.

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Oren Schuldiner for assistance and many helpful suggestions. The study was partially supported by the Alon Foundation (to N.B.).

Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Rambam Medical Center, Faculty of Medicine, The Technion, Haifa, Israel

    Joseph Itskovitz-Eldor, Dorit Karsenti & Michal Amit

  2. Department of Genetics, The Institute of Life Sciences, The Hebrew University, Jerusalem, Israel

    Maya Schuldiner, Amir Eden, Ofra Yanuka &  Nissim Benvenisty

  3. Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University, Jerusalem, Israel

    Hermona Soreq

Authors
  1. Joseph Itskovitz-Eldor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maya Schuldiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dorit Karsenti
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Amir Eden
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ofra Yanuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michal Amit
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hermona Soreq
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Nissim Benvenisty
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nissim Benvenisty.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D. et al. Differentiation of Human Embryonic Stem Cells into Embryoid Bodies Comprising the Three Embryonic Germ Layers. Mol Med 6, 88–95 (2000). https://doi.org/10.1007/BF03401776

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03401776

Keywords

Search

Navigation