Modeling Somitogenesis and Axial Development In Vitro with Axioloids

Axioloids are three-dimensional (3D) structures derived from pluripotent stem cells (PSCs) that model key aspects of human somitogenesis and early axis development in vitro. These mesoderm-based aggregates recapitulate essential morphogenetic features of the segmentation process, including axial elongation, sequential formation of epithelial somites with proper rostrocaudal patterning, and oscillatory activity of the segmentation clock. They further reproduce the spatiotemporal organization characteristic of early axial development, such as opposing FGF/WNT and retinoic acid signaling gradients and anteroposterior HOX gene expression patterns similar to those observed in vivo. Together, these attributes highlight axioloids as a robust and versatile platform for studying human axial development and congenital disorders of the spine and axial skeleton. In this chapter, we provide a concise, step-by-step protocol for generating axioloids, along with key developmental readouts and troubleshooting strategies to address common problems and issues encountered. This protocol provides a reproducible framework for producing and characterizing axioloids as an in vitro model of human axial development and disease.