Shaping 3D minimal model tissues with mechanical constraints to orchestrate muscle differentiation.

During development, biological tissues acquire their shape and organization by integrating internal and external cues, with mechanics playing a central role. Mechanical forces steer cell behavior and coordination, giving rise to self-organized architectures that underlie organ formation. While biochemical drivers of differentiation are well characterized, the contribution of topology and physical forces remains less understood. Here, we disentangle the role of alignment, tensile stress, and differentiation in three dimensions. Using self-organized aggregates of C2C12 myoblasts exposed to controlled stretching, we find that cells assemble into multilayered, actin-oriented tissues in which mechanical forces direct long-range 3D organization and promote myogenesis. Differentiation concentrates at the tissue core and surface, coinciding with regions of elevated stress and high cellular order. Single-molecule fluorescent hybridization confirms the overlap between differentiation hotspots and zones of strong alignment. These findings demonstrate that 3D alignment is a prerequisite for myoblast differentiation, and that mechanical constraints significantly boost its efficiency.