Three-dimensional stem cell models of mammalian gastrulation

Abstract

Gastrulation is a key milestone in the development of an organism. It is a period of cell proliferation and coordinated cellular rearrangement, that creates an outline of the body plan. Our current understanding of mammalian gastrulation has been improved by embryo culture, but there are still many open questions that are difficult to address because of the intrauterine development of the embryos and the low number of specimens. In the case of humans, there are additional difficulties associated with technical and ethical challenges. Over the last few years, pluripotent stem cell models are being developed that have the potential to become useful tools to understand the mammalian gastrulation. Here we review these models with a special emphasis on gastruloids and provide a survey of the methods to produce them robustly, their uses, relationship to embryos, and their prospects as well as their limitations.

Keywords: 3D models; embryonic stem cells; gastrulation; gastruloids; organoids.

FIGURE 1

General workflow for generating mouse and human gastruloids: (A) Mouse embryonic stem cells are maintained in conditions that maintain their pluripotency (e.g., ESL: serum and LIF; 2iL; 2iL+serum). Approximately 300 cells are seeded into 96‐well plates in the basal N2B27 medium. Aggregates are exposed to a 24 h pulse of CHIR between 48 and 72 h, and maintained in culture until 120 h. If extended culture is required, they can be “shaken” or embedded in Matrigel. (B) Prior to seeding, a subculture of human ESCs are plated in 6‐well plates from a single cell suspension in Nutristem for 96 h, after which the forming colonies are exposed to a pulse of CHIR for 24 h. Cells are then dissociated to single cells and plated in E6 medium containing CHIR and ROCKi for 24 h, followed by culture in E6 medium. (C) A number of critical factors that are important for both mouse and human gastruloids are listed. Batch‐tested components are essential, as is the quality and source of N2B27. A‐P indicates the anteroposterior axis.

FIGURE 2

Temporal and spatial reproducibility of Gastruloids. (A) A schematic example of gastruloids formed from Bra::GFP mouse embryonic stem cells over time. Note the early symmetry‐breaking, polarization of GFP, and the axial elongation over time. (B) Stereotypical polarization and patterning of specific markers in a typical 120 h gastruloids showing Tbxt (cyan), Sox2 (magenta), and a BMP signaling (yellow). (C) Typical brightfield images of E14Tg2A mouse ESCs at ∼120 h showing a high degree of reproducibility within a single experiment. Note the smooth elongating region (which would be TbxT positive).