Abstract Introduction Vascular smooth muscle cells (VSMCs) play a pivotal role in vascular homeostasis, with dysregulation leading to vascular complications. Human induced pluripotent stem cell (hiPSC)-derived VSMCs offer prospects for personalized disease modeling and regenerative strategies. Current research lacks comparative studies on the impact of 3D substrate properties under cyclic strain on phenotype adaptation in hiPSC-derived VSMCs. Here we investigated the potential of human mural cells derived from hiPSC-derived organoids (ODMCs) to undergo phenotypical adaptation under various biological and 3D mechanical stimuli. Methods and results ODMCs were cultured in 2D conditions with synthetic or contractile differentiation medium, or 3D Gelatin Methacryloyl (GelMa) substrates with varying degrees of functionalization and percentages to modulate material stiffness, elasticity, and crosslink density. Cells in 3D substrates were exposed to cyclic unidirectional strain. Phenotype characterization was conducted using specific markers through immunofluorescence and gene expression analysis. Under static 2D culture, ODMCs derived from hiPSCs exhibited a VSMC phenotype, expressing key mural markers, and demonstrated a level of phenotypic plasticity like primary human vSMCs. In static 3D culture, higher substrate stiffness, lower elasticity and higher crosslink density promoted a contractile phenotype in ODMCs and vSMCs. Dynamic stimulation in 3D substrate promoted a switch towards a contractile phenotype in both cell types. Conclusion Our study demonstrates a phenotypic plasticity of human ODMCs in response to 2D biological and 3D mechanical stimuli that equals that of primary human vSMCs. These findings may contribute to the advancement of tailored approaches for vascular disease modelling and regenerative strategies

The Effect of Mechanical Stimuli on the Phenotypic Plasticity of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells in a 3D Hydrogel