Reconstituting human somitogenesis in vitro.

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms, but remains largely elusive in humans due to ethical and technical limitations. Despite recent advances with pluripotent stem cell (PSC)-based approaches 1-5 , models that robustly recapitulate human somitogenesis in both space and time are still largely missing. Here, we introduce a PSC-derived mesoderm-based 3D model of human segmentation and somitogenesis, which we termed 'axioloid', that captures accurately the oscillatory dynamics of the segmentation clock and the morphological and molecular characteristics of sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF/WNT signaling gradients and retinoic acid (RA) signaling components. We identify an unexpected critical role of RA signaling in the stabilization of forming segments, indicating distinct, but also synergistic effects of RA and extracellular matrix (ECM) on the formation and epithelialization of somites. Importantly, comparative analysis demonstrates striking similarities of axioloids to the human embryo, further validated by the presence of a HOX code in axioloids. Lastly, we demonstrate the utility of axioloids to study the pathogenesis of human congenital spine diseases, by using patient-like iPSCs with mutations in HES7 and MESP2. These results suggest that axioloids represent a promising novel platform to study axial development and disease in humans.