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

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Abstract

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.

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References

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  3. Kubo, A., Shinozaki, K., & Shannon. (2004). Development of definitive endoderm from embryonic stem cells in culture. Development, 131(7), 1651–1662.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  13. Pyle, A. D., Lock, L. F., & Donovan, P. J. (2006). Neurotrophins mediate human embryonic stem cell survival. Nature Biotechnology, 24(3), 344–350.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.1.98.1.1.

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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).

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Authors and Affiliations

  1. Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium

    Dominika Dziedzicka, Christina Markouli, Lise Barbé, Claudia Spits, Karen Sermon & Mieke Geens

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  1. Dominika Dziedzicka
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  2. Christina Markouli
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  3. Lise Barbé
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  4. Claudia Spits
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Correspondence to Dominika Dziedzicka.

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Dziedzicka, D., Markouli, C., Barbé, L. et al. A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures. Stem Cell Rev and Rep 12, 721–730 (2016). https://doi.org/10.1007/s12015-016-9679-z

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