Stem Cell Reviews and Reports

, Volume 12, Issue 6, pp 721–730 | Cite as

A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures

  • Dominika DziedzickaEmail author
  • Christina Markouli
  • Lise Barbé
  • Claudia Spits
  • Karen Sermon
  • Mieke Geens


When aiming for homogenous embryoid body (EB) differentiation, the use of equal-sized EBs is required to avoid a size-induced differentiation bias. In this study we developed an efficient and standardized EB formation protocol for human pluripotent stem cells (hPSC) cultured in a laminin-521-based xeno-free system. As the cell proliferation rate of the cells growing on laminin-521 strongly affected the efficiency of aggregate formation, we found that recently passaged cells, as well as the addition of ROCK inhibitor, were essential for reproducible EB formation from hPSC single-cell suspensions. EBs could be obtained in a variety of differentiation media, in 96-well round-bottom plates and in hanging drops. Gene expression studies on differentially sized EBs from three individual human embryonic stem cell lines demonstrated that the medium used for differentiation influenced the differentiation outcome to a much greater extent than the number of cells used for the initial EB formation. Our findings give a new insight into factors that influence the EB formation and differentiation process. This optimized method allows us to easily manipulate EB formation and provide an excellent starting point for downstream EB-based differentiation protocols.


Human pluripotent stem cells Laminin-521 Embryoid body differentiation Forced aggregation Equal-sized embryoid bodies Differentiation 



We would like to thank A. Keller for proofreading the manuscript. This research was funded by the Methusalem grant of Vrije Universiteit Brussel granted to K.S. D.D. is a PhD fellow of Fund for Scientific Research – Flanders (Fonds voor Wetenschappelijk Onderzoek, FWO – Vlaanderen).

Compliance with Ethical Standards

Conflicts of Interest

The authors declare no potential conflicts of interest.

Supplementary material

12015_2016_9679_MOESM1_ESM.pdf (873 kb)
ESM 1 (PDF 872 kb)


  1. 1.
    Itskovitz-Eldor, J., Schuldiner, M., Karsenti, et al. (2000). Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Molecular Medicine, 6(2), 88–95.PubMedPubMedCentralGoogle Scholar
  2. 2.
    Takahashi, K., Tanabe, K., Ohnuki, et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131(5), 861–872.CrossRefPubMedGoogle Scholar
  3. 3.
    Kubo, A., Shinozaki, K., & Shannon. (2004). Development of definitive endoderm from embryonic stem cells in culture. Development, 131(7), 1651–1662.CrossRefPubMedGoogle Scholar
  4. 4.
    Elliott, D. A., Braam, S. R., Koutsis, K., et al. (2011). NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nature Methods, 8(12), 1037–1040.CrossRefPubMedGoogle Scholar
  5. 5.
    Lancaster, M. A., Renner, M., Martin, C.-A., et al. (2013). Cerebral organoids model human brain development and microcephaly. Nature, 501(7467), 373–379.CrossRefPubMedGoogle Scholar
  6. 6.
    Moon, S.-H., Ju, J., Park, S.-J., Bae, D., Chung, H.-M., & Lee, S.-H. (2014). Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies. Biomaterials, 35(23), 5987–5997.CrossRefPubMedGoogle Scholar
  7. 7.
    Ng, E. S., Davis, R. P., Azzola, L., Stanley, E. G., & Elefanty, A. G. (2005). Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood, 106(5), 1601–1603.CrossRefPubMedGoogle Scholar
  8. 8.
    Burridge, P. W., Anderson, D., Priddle, H., et al. (2007). Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells, 25(4), 929–938.CrossRefPubMedGoogle Scholar
  9. 9.
    Hwang, Y.-S., Chung, B. G., Ortmann, D., Hattori, N., Moeller, H.-C., & Khademhosseini, A. (2009). Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11. Proceedings of the National Academy of Sciences of the United States of America, 106(40), 16978–16983.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Mohr, J. C., Zhang, J., Azarin, S. M., et al. (2010). The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells. Biomaterials, 31(7), 1885–1893.CrossRefPubMedGoogle Scholar
  11. 11.
    Valamehr, B., & Jonas, S. (2008). Hydrophobic surfaces for enhanced differentiation of embryonic stem cell-derived embryoid bodies. Proceedings of the National Academy of Sciences of the United States of America, 105, 14459–14464.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Amit, M., Carpenter, M. K., Inokuma, M. S., et al. (2000). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Developmental Biology, 227(2), 271–278.CrossRefPubMedGoogle Scholar
  13. 13.
    Pyle, A. D., Lock, L. F., & Donovan, P. J. (2006). Neurotrophins mediate human embryonic stem cell survival. Nature Biotechnology, 24(3), 344–350.CrossRefPubMedGoogle Scholar
  14. 14.
    Chen, G., Hou, Z., Gulbranson, D. R., & Thomson, J. A. (2010). Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell, 7(2), 240–248.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ohgushi, M., Matsumura, M., Eiraku, M., et al. (2010). Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell, 7(2), 225–239.CrossRefPubMedGoogle Scholar
  16. 16.
    Watanabe, K., Ueno, M., Kamiya, D., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology, 25(6), 681–686.CrossRefPubMedGoogle Scholar
  17. 17.
    Rodin, S., Antonsson, L., Niaudet, C., et al. (2014). Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nature Communications, 5, 3195.CrossRefPubMedGoogle Scholar
  18. 18.
    Rodin, S., Antonsson, L., Hovatta, O., & Tryggvason, K. (2014). Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions. Nature Protocols, 9(10), 2354–2368.CrossRefPubMedGoogle Scholar
  19. 19.
    Ungrin, M. D., Joshi, C., Nica, A., Bauwens, C., & Zandstra, P. W. (2008). Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates. PloS One, 3(2), e1565.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Mateizel, I., De Temmerman, N., Ullmann, U., et al. (2006). Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Human Reproduction, 21(2), 503–511.CrossRefPubMedGoogle Scholar
  21. 21.
    Mateizel, I., Spits, C., De Rycke, M., Liebaers, I., & Sermon, K. (2010). Derivation, culture, and characterization of VUB hESC lines. In Vitro Cellular & Developmental Biology - Animal, 46(3-4), 300–308.CrossRefGoogle Scholar
  22. 22.
    Jacobs, K., Mertzanidou, A., Geens, M., Thi Nguyen, H., Staessen, C., & Spits, C. (2014). Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations. Nature Communications, 5(May), 4227.PubMedGoogle Scholar
  23. 23.
    Ng, E. S., Davis, R., Stanley, E. G., & Elefanty, A. G. (2008). A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nature Protocols, 3(5), 768–776.CrossRefPubMedGoogle Scholar
  24. 24.
    Burridge, P. W., Thompson, S., Millrod, M. A., et al. (2011). A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PloS One, 6(4), e18293.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Lin, Y., Chen, G., Engineering, G., & Facility, C. (2014). Embryoid body formation from human pluripotent stem cells in chemically defined E8 media, 1–4. doi: 10.3824/stembook.
  26. 26.
    Beers, J., Gulbranson, D. R., George, N., et al. (2012). Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nature Protocols, 7(11), 2029–2040.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Pettinato, G., Vanden Berg-Foels, W. S., Zhang, N., & Wen, X. (2014). ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells. PloS One, 9(11), e100742.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Stover, A. E., & Schwartz, P. H. (2011). The generation of embryoid bodies from feeder-based or feeder-free human pluripotent stem cell cultures. Methods in Molecular Biology, 767, 391–398.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Jacobs, K., Zambelli, F., Mertzanidou, A., et al. (2016). Higher-density culture in human embryonic stem cells results in DNA damage and genome instability. Stem Cell Reports, 6(3), 330–341.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Pettinato, G., Wen, X., & Zhang, N. (2014). Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays. Scientific Reports, 4, 7402.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Yirme, G., Amit, M., Laevsky, I., Osenberg, S., & Itskovitz-Eldor, J. (2008). Establishing a dynamic process for the formation, propagation, and differentiation of human embryoid bodies. Stem Cells and Development, 17, 1227–1241.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Dominika Dziedzicka
    • 1
    Email author
  • Christina Markouli
    • 1
  • Lise Barbé
    • 1
  • Claudia Spits
    • 1
  • Karen Sermon
    • 1
  • Mieke Geens
    • 1
  1. 1.Research Group Reproduction and Genetics, Faculty of Medicine and PharmacyVrije Universiteit BrusselBrusselsBelgium

Personalised recommendations