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Method Article
Generating gastruloids with somite-like structures from mouse embryonic stem cells
Susanne Carina van den Brink
Hubrecht Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3683-7737
Alexander van Oudenaarden
Hubrecht Institute
Corresponding Author
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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
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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.
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This is a preprint. It has not completed peer review.
Method Article
Generating gastruloids with somite-like structures from mouse embryonic stem cells
Susanne Carina van den Brink
Hubrecht Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3683-7737
Alexander van Oudenaarden
Hubrecht Institute
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
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.
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.