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