PROTOCOLS

References and Links to Protocols, mostly located on Protocol Exchange. Do get in touch if you want to discuss any experimental details or techniques further.

GENERATING HUMAN GASTRULOIDS

Gastruloids are aggregates of Pluripotent Stem Cells (PSCs) which, when exposed to differentiation medium and plated within defined conditions, undergo trilineage differentiation to all three germ layers (mesoderm, ectoderm and endoderm) with constitutive cell types organised spatiotemporally along 3 axes (Becarri et. al. 2018). They also undergo morphological shape changes including axial elongation through convergent extension cell movements. The gastruloid method has been well-established using mouse Embryonic Stem Cells (mESCs) and mouse induced PSCs (iPSCs) (van den Brink et. al., 2014; Turner et. al. 2016a; Turner et. al. 2016b; Bailie-Johnson et. al. 2015; Turner et. al. 2017a; Turner et. al. 2017b, Beccari et. al., 2018, See method in Girgin et. al., 2018). Here, we describe a new method to generate equivalent gastruloids from human pluripotent stem cells (hPSCs). hPSCs are able to generate axially elongated human gastruloids with evidence of spatially organised germ layers and comparable features to their mouse counterparts (Moris et. al., 2020).

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GENERATING MOUSE GASTRULOIDS

Mouse embryonic stem cells (mESCs) can form all of the tissues of the developing embryo in vivo; yet they develop into spatially disorganised structures when grown in vitro as embryoid bodies. Here, we present a 5-7 day protocol for generating aggregates of mESCs, that we have dubbed gastruloids, that mimic the early post-implantation development of the embryo by virtue of their defined, small size. When cultured in the appropriate conditions, gastruloids break radial symmetry, polarise their gene expression, and specify the major body axes. They further undergo a gastrulation-like process and axial elongation, and follow a temporal programme of gene expression that corresponds to post-occipital embryonic development, exemplified by the collinear expression of Hox genes along the developing anteroposterior axis. A key strength of this system is the ability to reproducibly and robustly generate large numbers of gastruloids under defined conditions that will enable demanding experimental approaches, normally very difficult or impossible to accomplish with embryonic material.

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GENERATING MOUSE GASTRULOIDS WITH SOMITE-LIKE STRUCTURES

Gastruloids are aggregates of mouse embryonic stem cells that can be used to study key aspects of mammalian post-implantation development in vitro. Gastruloids generated with previously published protocols do not generate somite-like structures. Here, we describe a modified version of the gastruloids culture protocol that results in gastruloids that do generate somite-like structures in vitro (van den Brink et al., Nature, 2020). Under these conditions, about 50% of the gastruloids generated form structures with features that are characteristic of somites.
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.

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