Derivation of axioloids, an in vitro model of somitogenesis and axial development from human pluripotent stem cells

Abstract Here we describe a step-by-step protocol for the derivation of axioloids, a pluripotent stem cell (PSC)-based 3D in vitro model of human segmentation and somitogenesis. Mesoderm-based axioloids capture reproducibly core features of the segmentation process in vitro including axial elongation, sequential formation of properly patterned epithelial somites and coordinated oscillatory activity of the segmentation clock. Axioloids recapitulate furthermore key morphogenetic and molecular features of post-gastrulation human embryos, including temporally coordinated emergence and spatial organization of somitogenesis-associated mesodermal cell populations, presence of opposing FGF/WNT and RA signaling gradients and spatiotemporally controlled expression of HOX genes. In addition to increasing our still limited understanding of human axial development, axioloids can be used to model and study congenital diseases of the human spine, when utilizing patient-like iPSCs with defined pathogenic mutations in genes such as HES7 or MESP2.