Generation of Mouse Pluripotent Stem Cell-derived Trunk-like Structures: An in vitro Model of Post-implantation Embryogenesis

Abstract

Post-implantation mammalian embryogenesis involves profound molecular, cellular, and morphogenetic changes. The study of these highly dynamic processes is complicated by the limited accessibility of in utero development. In recent years, several complementary in vitro systems comprising self-organized assemblies of mouse embryonic stem cells, such as gastruloids, have been reported. We recently demonstrated that the morphogenetic potential of gastruloids can be further unlocked by the addition of a low percentage of Matrigel as an extracellular matrix surrogate. This resulted in the formation of highly organized trunk-like structures (TLSs) with a neural tube that is frequently flanked by bilateral somites. Notably, development at the molecular and morphogenetic levels is highly reminiscent of the natural embryo. To facilitate access to this powerful model, here we provide a detailed step-by-step protocol that should allow any lab with access to standard cell culture techniques to implement the culture system. This will provide the user with a means to investigate early mid-gestational mouse embryogenesis at an unprecedented spatiotemporal resolution.

Keywords: Embryoids; Gastrulation; Gastruloids; In vitro models; Morphogenesis; Organoids; Self-organization; Somites; Stem cells; Trunk-like structures.

Figure 1.. Optimal embryonic stem cell culture densities for successful TLS generation.

A. mESC culture densities suitable for TLS generation (24 h and 48 h after seeding). B. mESC culture density unsuitable for TLS generation (96 h after seeding). Scale bars for all panels, 50 μm.

Figure 2.. Schematic overview of the TLS generation protocol.

Workflow for the generation of trunk-like-structures (TLS) from seeding of MEFs to downstream analysis. MG, Matrigel; CL, CHIR+LDN; MEFs, mouse embryonic fibroblasts; mESCs, mouse embryonic stem cells.

Figure 3.. MEF depletion prior to mESC aggregation.

MEFs attach to the 0.1% gelatin-coated wells. Scale bars, 50 μm.

Figure 4.. Examples of expected morphology of mESC-derived aggregates at several timepoints during TLS generation.

A, B. mESC-derived aggregates at 48 h and 72 h after aggregation are round without clear signs of symmetry breaking. C. At 96 h after aggregation, the structures have clearly broken symmetry and are teardrop shaped. The white arrowheads indicate the posterior pole, where localized expression of Brachyury is expected. Note that, depending on the cell line used, aggregates may establish the teardrop-like morphology prior to 96 h. In that case, structures should be embedded in Matrigel earlier (as soon as the teardrop-like morphology is observed) to achieve optimal TLS efficiency (see main text for details). D. Upon addition of 5% Matrigel, the aggregates will establish an architecture reminiscent of the embryonic trunk, with somites (magenta arrowheads) flanking a neural tube (green arrowheads). Chemical modulation with a WNT agonist (5 μM CHIR99021) and BMP inhibitor (600 nM LDN193189) results in compromised neural tube development and formation of excess somites arranged like a “bunch-of-grapes” (TLS^CL). Scale bars for all panels, 50 μm. A, Anterior; P, Posterior.

Figure 5.. Schematic representation of plate positioning for media changes during the TLS generation procedure.

The plate is tilted at a 30-40° angle on a clean bench and the media is carefully aspirated with a multichannel pipet, avoiding disturbance of the aggregates.

Figure 6.. Expected outcome of the TLS protocol.

A. Examples of trunk-like structures (TLSs) 24 h after addition of 5% Matrigel (total culture time 120 h). The left upper structure is immunostained with a SOX2 antibody and counterstained with DAPI, labeling the neural tube and nuclei, respectively. Somites are indicated with magenta arrowheads or with S1, S2, etc. (in magnifications); S1-S8, Somite 1-Somite 8. Neural tube (NT) is indicated with a green arrowhead. A, Anterior; P, Posterior. B. Expected outcome of TLSs subjected to chemical modulation (TLS^CL) is compromised neural tube development and formation of excess somites arranged like a “bunch-of-grapes.” Somites are indicated with magenta arrowheads. Scale bars, 100 μm (whole structure) or 20 μm (magnifications). A, Anterior; P, Posterior.