Robust self-organization of livestock pluripotent stem cells into post-gastrulation embryo models with advanced neuronal and mesodermal structures

Mammalian body plan formation arises from the self-organization of pluripotent cells through conserved morphogenetic processes that are difficult to study in vivo. Stem cell-based embryo models (SEMs) offer accessible three-dimensional systems to investigate these events but are currently limited to mouse and human cells and largely recapitulate posterior embryonic structures. In addition, no in-vitro models exist for post-gastrulation development in ungulate species, whose early development differs from that of rodents and primates. Here, we establish SEMs for two common ungulates, sheep and pig, using pluripotent stem cell-derived aggregates. We generate ovine and porcine gastruloids that recapitulate key features of gastrulation, including germ layer specification, symmetry breaking, and axial elongation. We further develop ovine trunk-like structures (oTLSs) that robustly model post-gastrulation trunk development, exhibiting sustained elongation, neuromesodermal progenitor maintenance, segmented somite formation, and a central neural tube-like axis. Time-resolved single-cell RNA sequencing combined with immunostaining reveals coordinated emergence of neural, mesodermal, and intermediate mesodermal lineages arranged along an anteroposterior axis. Notably, oTLSs generate dorsal neural derivatives, anterior neuronal populations, and renal primordia, representing an expansion in the lineage repertoire reported for existing trunk models. Together, this work extends SEMs to livestock species and establishes a platform for comparative mammalian developmental studies, with potential applications in fundamental research, veterinary toxicology, and agricultural biotechnology.