Stem Cell Reviews and Reports

, Volume 5, Issue 4, pp 410–419 | Cite as

A Three Dimensional Anchorage Independent In Vitro System for the Prolonged Growth of Embryoid Bodies to Study Cancer Cell Behaviour and Anticancer Agents

  • Chui-Yee Fong
  • Li-Ling Chak
  • Arjunan Subramanian
  • Jee-Hian Tan
  • Arijit Biswas
  • Kalamegam Gauthaman
  • Mahesh Choolani
  • Woon-Khiong Chan
  • Ariff Bongso


We describe a three dimensional (3D) anchorage independent in vitro protocol for the prolonged growth of human embryoid bodies (EBs) up to 90 days. We grew hESCs (46XX) in methylcellulose (MC) in motion culture in the presence of EB medium (EB), EB medium with Matrigel (EB + MAT), bulk culture medium (BCM), and BCM medium with Matrigel (BCM + MAT). All four experimental groups produced embryoid bodies (EBs) which with prolonged growth to 90 days acquired blood vessels and tissues from all three germ layers. Based on histology, microarray gene expression profiles and the definition for experimental teratomas, we could classify the EBs into early EBs, mature EBs and teratomas. The EB + MAT group produced the highest number of teratomas and their microarray data suggested the presence of inductive microenvironment niches and activation of pathways for self-organization, morphogenesis and growth. When we microinjected hepatocarcinoma-Green Fluorescent Protein cells (HepG2-GFP) (46XY) into the teratomas, after 10 days the HepG2-GFP cells had grown inside the teratoma as confirmed by confocal microscopy and SRY gene analysis. This 3D-MC-(EB + MAT) in vitro system requires few cells to produce many teratomas, can be used to test pluripotency of potential human embryonic and induced pluripotent stem cell lines (hESC, hiPSC), and is an experimental humanized platform to study cancer cell behavior.


Embryoid bodies Histology Human embryonic stem cells In vitro teratoma assay Microarray Microinjection 



The authors thank Dr Gan Shu Uin (National University of Singapore) for a gift of the HepG2-GFP cells. This project was supported by funds from the National University of Singapore (R-174-000-089-133) and the National Medical Research Council, Singapore (R-174-000-103-213).


  1. 1.
    Shih, C. Forman, S. J. Chu, P. & Slovak, M. (2007). Human embryonic stem cells are prone to generate primitive, undifferentiated tumors in engrafted human fetal tissues in severe combined immunodeficient mice. Stem Cells & Development, 16, 893–902.CrossRefGoogle Scholar
  2. 2.
    Prokhorova, T. A. Harkness, L. M. Frandsen, U. Ditzel, N. Schrøder, H. D. Burns, J. S. et al. (2009). Teratoma formation by human embryonic stem cells is site-dependent and enhanced by the presence of Matrigel. Stem Cells and Development, 18, 47–54.CrossRefPubMedGoogle Scholar
  3. 3.
    Takahashi, K. Tanabe, K. Ohnuki, M. Narita, M. Ichisaka, T. Tomoda, K. et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872.CrossRefPubMedGoogle Scholar
  4. 4.
    Blum, B. & Benvenisty, N. (2008). The tumorigenicity of human embryonic stem cells. Advanced Cancer Research, 100, 133–158.CrossRefGoogle Scholar
  5. 5.
    Brivanlou, A. H. Gage, F. H. Jaenisch, R. Jessell, T. Melton, D. & Rossant, J. (2003). Setting standards for human embryonic stem cells. Science, 300, 913–916.CrossRefPubMedGoogle Scholar
  6. 6.
    Tzukerman, M. Rosenberg, T. Ravel, Y. Reiter, I. Coleman, R. & Skorecki, K. (2003). An experimental platform for studying growth and invasiveness of tumor cells within teratomas derived from human embryonic stem cells. Proceedings of the National Academy of Sciences USA, 100, 13507–13512.CrossRefGoogle Scholar
  7. 7.
    Tzukerman, M. Rosenberg, T. Reiter, I. Eliezer, S. B. Denkberg, G. Coleman, R. et al. (2006). The influence of a human embryonic stem cell-derived microenvironment on targeting of human solid tumor xenografts. Cancer Research, 66, 3792–3801.CrossRefPubMedGoogle Scholar
  8. 8.
    Lensch, M. W. Schlaeger, T. M. Zon, L. I. & Daley, G. Q. (2007). Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera. Cell Stem Cell, 1, 253–258.CrossRefPubMedGoogle Scholar
  9. 9.
    Aleckovic, M. & Simon, C. (2008). Is teratoma formation in stem cell research a characterization tool or a window to developmental biology? Reproductive Biomedicine Online, 17, 270–280.PubMedCrossRefGoogle Scholar
  10. 10.
    Itskovitz-Eldor, J. Schuldiner, M. Karsenti, D. Eden, A. Yanuka, O. Amit, M. et al. (2000). Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Molecular Medicine, 6, 88–95.PubMedGoogle Scholar
  11. 11.
    Karbanova, J. & Mokry, J. (2002). Histological and histochemical analysis of embryoid bodies. Acta Histochemistry, 104, 361–365.CrossRefGoogle Scholar
  12. 12.
    Rust, W. L. Sadasivam, A. & Dunn, N. R. (2006). Three-dimensional extracellular matrix stimulates gastrulation-like events in human embryoid bodies. Stem Cells and Development, 15, 889–904.CrossRefPubMedGoogle Scholar
  13. 13.
    Cameron, C. M. Hu, W. S. & Kaufman, D. S. (2006). Improved development of human embryonic stem cell-derived embryoid bodies by stirred vessel cultivation. Biotechnology and Bioengineering, 94, 938–948.CrossRefPubMedGoogle Scholar
  14. 14.
    Du, P. Kibbe, W. A. & Lin, S. M. (2008). Lumi: a pipeline for processing Illumina microarray. Bioinformatics, 24, 1547–1548.CrossRefPubMedGoogle Scholar
  15. 15.
    Lin, S. M. Du, P. Huber, W. & Kibbe, W. A. (2008). Model-based variance-stabilizing transformation for Illumina microarray data. Nucleic Acids Research, 36, e11.CrossRefPubMedGoogle Scholar
  16. 16.
    Sturn, A. Quackenbush, J. & Trajanoski, Z. (2002). Genesis: cluster analysis of microarray data. Bioinformatics, 18, 207–208.CrossRefPubMedGoogle Scholar
  17. 17.
    Huang, D. W. Sherman, B. T. & Lempicki, R. A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols, 4, 44–57.CrossRefGoogle Scholar
  18. 18.
    Liu, X. Yu, X. Zack, D. J. Zhu, H. & Qian, J. (2008). TiGER: a database for tissue-specific gene expression and regulation. BMC Bioinformatics, 9, 271–275.CrossRefPubMedGoogle Scholar
  19. 19.
    Pirooznia, M. Nagarajan, V. & Deng, Y. (2007). GeneVenn: a web application for comparing gene lists using Venn diagrams. Bioinformation, 1, 420–422.PubMedGoogle Scholar
  20. 20.
    ten Berge, D. Koole, W. Fuerer, C. Fish, M. Eroglu, E. & Nusse, R. (2008). Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell, 3, 508–518.CrossRefPubMedGoogle Scholar
  21. 21.
    Blum, B. Bar-Nur, O. Golan-Lev, T. & Benvenisty, N. (2009). The anti-apoptotic gene survivin contributes to teratoma formation by human embryonic stem cells. Nature Biotechology, 27, 281–287.CrossRefGoogle Scholar
  22. 22.
    Garner, E. & Raj, K. (2008). Protective mechanisms of p53–p21-pRb proteins against DNA damage-induced cell death. Cell Cycle, 7, 277–282.PubMedGoogle Scholar
  23. 23.
    Croucher, D. R. Saunders, D. N. Lobov, S. & Ranson, M. (2008). Revisiting the biological roles of PAI2 (SERPINB2) in cancer. Nature Review Cancer, 8, 535–545.CrossRefGoogle Scholar
  24. 24.
    Wu, M. X. (2003). Roles of the stress-induced gene IEX-1 in regulation of cell death and oncogenesis. Apoptosis, 8, 11–18.CrossRefPubMedGoogle Scholar
  25. 25.
    Ben-Porath, I. Thomson, M. W. Carey, V. J. Ge, R. Bell, G. W. & Regev, A. (2008). An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nature Genetics, 40, 499–507.CrossRefPubMedGoogle Scholar
  26. 26.
    Kleinman, H. K. & Martin, G. R. (2005). Matrigel: basement membrane matrix with biological activity. Seminars in Cancer Biology, 15, 378–386.CrossRefPubMedGoogle Scholar
  27. 27.
    Philip, D. Chen, S. S. Fitzgerald, W. Orenstein, J. Margolis, L. & Kleinman, H. (2005). Complex extracellular matrices promote tissue-specific stem cell differentiation. Stem Cells, 23, 288–296.CrossRefGoogle Scholar
  28. 28.
    Reubinoff, B. E. Pera, M. F. Fong, C. Y. Trounson, A. & Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vivo. Nature Biotechology, 18, 399–404.CrossRefGoogle Scholar
  29. 29.
    Mauck, R. L. Li, W. J. & Tuan, R. S. (2009). Microenvironmental determinants of stem cell fate. In U. Meyer, J. Handschel, H. P. Weismann & T. Meyer (Eds.), Fundamentals of tissue engineering and regenerative medicine. Berlin: Springer.Google Scholar

Copyright information

© Springer Science + Business Media 2009

Authors and Affiliations

  • Chui-Yee Fong
    • 1
  • Li-Ling Chak
    • 2
  • Arjunan Subramanian
    • 1
  • Jee-Hian Tan
    • 2
  • Arijit Biswas
    • 1
  • Kalamegam Gauthaman
    • 1
  • Mahesh Choolani
    • 1
  • Woon-Khiong Chan
    • 2
  • Ariff Bongso
    • 1
  1. 1.Department of Obstetrics & Gynecology, Yong Loo Lin School of MedicineNational University of SingaporeKent RidgeSingapore
  2. 2.Department of Biological SciencesNational University of SingaporeKent RidgeSingapore

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