title: A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures.
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.
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium. dominika.dziedzicka@vub.ac.be.
authors.forename: Dominika
authors.initials: D
authors.lastname: Dziedzicka
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
authors.forename: Christina
authors.initials: C
authors.lastname: Markouli
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
authors.forename: Lise
authors.initials: L
authors.lastname: Barbé
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
authors.forename: Claudia
authors.initials: C
authors.lastname: Spits
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Sermon
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
authors.forename: Mieke
authors.initials: M
authors.lastname: Geens
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D056945:Hep G2 Cells; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000494:pharmacology*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D007797:Laminin; C474559:laminin 11 protein, human
keywords: Differentiation; Embryoid body differentiation; Equal-sized embryoid bodies; Forced aggregation; Human pluripotent stem cells; Laminin-521