Gastruloids: Pluripotent stem cell models of mammalian gastrulation and embryo engineering

Abstract

Gastrulation is a fundamental and critical process of animal development whereby the mass of cells that results from the proliferation of the zygote transforms itself into a recognizable outline of an organism. The last few years have seen the emergence of a number of experimental models of early mammalian embryogenesis based on Embryonic Stem (ES) cells. One of this is the Gastruloid model. Gastruloids are aggregates of defined numbers of ES cells that, under defined culture conditions, undergo controlled proliferation, symmetry breaking, and the specification of all three germ layers characteristic of vertebrate embryos, and their derivatives. However, they lack brain structures and, surprisingly, reveal a disconnect between cell type specific gene expression and tissue morphogenesis, for example during somitogenesis. Gastruloids have been derived from mouse and human ES cells and several variations of the original model have emerged that reveal a hereto unknown modularity of mammalian embryos. We discuss the organization and development of gastruloids in the context of the embryonic stages that they represent, pointing out similarities and differences between the two. We also point out their potential as a reproducible, scalable and searchable experimental system and highlight some questions posed by the current menagerie of gastruloids.

Keywords: Embryos; Experimental embryology; Gastruloids; Systems biology.

Fig. 1.. Schematic overview of mouse gastrulation.

(A) Mouse embryo development from fertilisation through implantation, and the establishment of the body plan through gastrulation. (B) Signalling gradients and biases from the extraembryonic ectoderm and visceral endoderm set up the localisation of the Primitive Streak in the proximal-posterior epiblast. T, Brachyury. (C) The ordered emergence of mesoderm from the primitive streak, in both a proximodistal and circumnavigation from posterior-to-anterior. Al, Allantois; YS, Yolk Sac; PGCs, Primordial Germ Cells; CM, Cardiac Mesoderm; LPM, Lateral Plate Mesoderm; IM, Intermediate Mesoderm; PM, Paraxial Mesoderm; AM, Axial Mesoderm.

Fig. 2.. Making Gastruloids.

(A, B) Schematic protocol for generating mouse and human gastruloids. Pluripotent stem cells are dissociated to a single cell suspension, and an particular cell number (typically 300–500 cells) are added to each well of a 96-well plate with rounded bottoms (A). The cells will settle and aggregate to form a 3D spherical structure that, over time, begins to break symmetry, polarize its gene expression, and undergo axial elongation. The timing of exposure to agonists of Wnt signalling differs for mouse and human ES cells (B). (C) Representative microscopy images of a developing mouse gastruloid timeline between about 72 and 126 h after aggregation. Images from a light sheet movie of a gastruloid bearing ubiquitously expressed GPI linked GFP (U.Fiuza unpublished). Scale bar: 100 μm. (D, E) Sample ollections of 120m hrs old gastruloids from E14 ES (D) and P19 embryo carcinoma (E) cells.

Fig. 3.. Relationship between mouse embryo (A) and gastruloid (B) development.

Gastruloids exhibit the emergence and polarization of Bra and Nodal gene expression in the extending domain.

Fig. 4.. Relationship between different embryo-like models and gastruloids.

Pictorial diagram of the relationship between various stem cell-based embryo-like models and the 3D gastruloids. ESC, Embryonic Stem Cells; TS, Trophoblast Stem cells; XEN,; Chi, Chiron; Epi, Epiblast-like Stem Cells; Mtg, Matrigel; WNTi, WNT signalling inhibition.

Fig. 5.. Relationship between mouse embryos and gastruloids.

Outline of a mouse embryo at E8.5 with axial organization and germ layer derivatives indicated (A) compared to a 120 h old gastruloid (B and see text for details). (C) Examples of patterns of gene expression in 120 hrs old gastruloids. TLC2 is a reporter for canonical Wnt signalling.

Fig. 6.. Human embryos and human gastruloids.

Comparison of human embryogenesis and human gastruloid development (see text for details).