Abstract
Gastruloids are embryonic organoids made from small, defined numbers of mouse embryonic stem cells (mESCs) aggregated in suspension culture, which over time form 3D structures that mimic many of the features of early mammalian development. Unlike embryoid bodies that are usually disorganized when grown over several days, gastruloids display distinct, well-organized gene expression domains demarcating the emergence of the three body axes, anteroposterior axial elongation, and implementation of collinear Hox transcriptional patterns over 5-7 days of culture. As such gastruloids represent a useful experimental system that is complementary to in vivo approaches in studying early developmental patterning mechanisms regulating the acquisition of cell fates. In this protocol, we describe the most recent method for generating gastruloids with high reproducibility, and provide a comprehensive list of possible challenges as well as steps for protocol optimization.