Generation of Human Elongating Multi-Lineage Organized (EMLO) Gastruloids

Abstract The inaccessibility of scientific discovery to human development at post-implantation, including specialization and differentiation, necessitates sophisticated heterogenous 3D in vitro models. This protocol describes the generation and self-organization of human elongating multi-lineage organized (EMLO) gastruloids in suspension without the need for supplied extracellular matrix (ECM). EMLO gastruloids can be matured to large organoid states comprised of interconnected tissue microstructures of the trunk. These include correlates of the neuromesodermal progenitor-derived central nervous system, the neural crest cell-derived peripheral nervous system, the endoderm-derived primitive gut tube, and the mesoderm-derived splanchnic mesenchyme. Herein, we provide critical details for the reproducible generation of human EMLOs in vitro.