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Method Article
Susanne Carina van den Brink
Hubrecht Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3683-7737
Alexander van Oudenaarden
Hubrecht Institute
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Gastruloids are aggregates of mouse embryonic stem cells that can be used to study key aspects of mammalian post-implantation development in vitro1–4. Gastruloids generated with previously published protocols do not generate somite-like structures4–6. Here, we describe a modified version of the gastruloids culture protocol5,6 that results in gastruloids that do generate somite-like structures in vitro (van den Brink et al., Nature, 2020)7. Under these conditions, about 50% of the gastruloids generated form structures with features that are characteristic of somites7.
This protocol takes 6 days, with relatively little hands-on time. The protocol starts with the aggregation of the cultured cells. Then, the Wnt-agonist Chiron is added 2 days (48h) later. The medium of the aggregates is replaced 3 days (72h) after aggregation. To induce somite-formation, gastruloids are embedded in Matrigel 4 days (96h) after aggregation. After 5 days (120h) of culture, gastruloids resemble E8.5 mouse embryos. At this timepoint they can be fixed (fixative is added on day 5 and washed away on day 6 after overnight incubation in PFA) to prepare them for staining or microscopy experiments.
Keywords
Gastruloids, mouse embryonic stem cells, Matrigel, somites
The authors declare no competing interests.
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Associated Publications
10.1038/s41586-020-2024-3
- This protocol has been used in the following primary research paper: Title: Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids Authors: Susanne C. van den Brink, Anna Alemany, Vincent van Batenburg, Naomi Moris, Marloes Blotenburg, Judith Vivié, Peter Baillie-Johnson, Jennifer Nichols, Katharina F. Sonnen, Alfonso Martinez Arias & Alexander van Oudenaarden Journal: Nature. Status: in press. Expected publication date: 2020.
Method Article
Susanne Carina van den Brink
Hubrecht Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3683-7737
Alexander van Oudenaarden
Hubrecht Institute
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 19 Feb, 2020
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Gastruloids are aggregates of mouse embryonic stem cells that can be used to study key aspects of mammalian post-implantation development in vitro1–4. Gastruloids generated with previously published protocols do not generate somite-like structures4–6. Here, we describe a modified version of the gastruloids culture protocol5,6 that results in gastruloids that do generate somite-like structures in vitro (van den Brink et al., Nature, 2020)7. Under these conditions, about 50% of the gastruloids generated form structures with features that are characteristic of somites7.
This protocol takes 6 days, with relatively little hands-on time. The protocol starts with the aggregation of the cultured cells. Then, the Wnt-agonist Chiron is added 2 days (48h) later. The medium of the aggregates is replaced 3 days (72h) after aggregation. To induce somite-formation, gastruloids are embedded in Matrigel 4 days (96h) after aggregation. After 5 days (120h) of culture, gastruloids resemble E8.5 mouse embryos. At this timepoint they can be fixed (fixative is added on day 5 and washed away on day 6 after overnight incubation in PFA) to prepare them for staining or microscopy experiments.
Loading...
Associated Publications
10.1038/s41586-020-2024-3
- This protocol has been used in the following primary research paper: Title: Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids Authors: Susanne C. van den Brink, Anna Alemany, Vincent van Batenburg, Naomi Moris, Marloes Blotenburg, Judith Vivié, Peter Baillie-Johnson, Jennifer Nichols, Katharina F. Sonnen, Alfonso Martinez Arias & Alexander van Oudenaarden Journal: Nature. Status: in press. Expected publication date: 2020.