Single-cell spatial mapping reveals reproducible cell type organization and spatially-dependent gene expression in gastruloids.

Gastruloids are three-dimensional stem-cell-based models that recapitulate key aspects of mammalian gastrulation, including formation of an anterior-posterior (AP) axis. However, we do not have detailed spatial information about gene expression and cell type organization, particularly at the level of individual gastruloids. Here, we report a spatially resolved, single-cell molecular catalog of the transcriptomes of 26 individual gastruloids. We found that cell type composition and spatial organization were remarkably consistent across gastruloids. Posterior cell types formed distinct, organized clusters, while anterior cell types were more disorganized. To distinguish progressive differentiation from cell type differences, we developed the L-metric, a parameter-free quantification of mutually exclusive gene expression. This analysis revealed spatial organization without explicit encoding, recapitulated known cell type relationships, and identified novel gene expression states and spatial subclusters within cell types. We confirmed that in gastruloids, NMP differentiation occurred through a continuous, spatially-coordinated process. We also showed that endothelial precursors exhibited unique spatial organization and had distinct gene expression profiles dependent on their association with anterior somitic or posterior endodermal tissues. This work enables the rigorous use of gastruloids as models for studying the molecular mechanisms underlying mammalian development and tissue organization, and introduces novel computational tools for analyzing spatially-resolved single-cell datasets.