Cytoskeletal and Extracellular Factors Regulating Stem Cell Differentiation: Mechanisms, Tools, and Emerging Applications

Abstract Stem cell lineage determination is governed not only by biochemical cues but also by the organization and force-generating state of the cytoskeleton, which integrates extracellular mechanical cues into transcriptional and epigenetic responses. Cytoskeletal organization and remodeling play essential roles in stem cell fate determination, as well as other relevant biological processes including development, tissue regeneration, and disease progression. In this review, we first outline the different types of cytoskeletal structures—actin filaments, microtubules, and intermediate filaments—and their coordinated roles in cell shape, polarity, and contractility. We then discuss how extracellular cues, including matrix stiffness, geometry, topography, shear, stretch, and compression remodel cytoskeletal distribution and adhesion dynamics to affect stem cell lineage commitment. We highlight matrix viscoelasticity and stress relaxation as time-dependent mechanical regulators that modulate traction transmission, focal adhesion, and cytoskeletal tension, thereby directing stem cell differentiation outcomes beyond stiffness alone. We next discuss mechanotransduction pathways, such as RhoA/ROCK-mediated contractility, Wnt/β-catenin, Piezo1/Piezo2, and YAP/TAZ nuclear shuttling, which couple cytoskeletal forces to gene regulation. We further summarize quantitative approaches to measure cell molecular forces, including traction force microscopy, molecular tension sensors, and advanced live-cell imaging platforms. Finally, we discuss the translational potential of cytoskeleton-informed design principles in regenerative medicine, where they can be leveraged to improve stem cell biomanufacturing, optimize tunable biomaterials, and develop more predictive mechanopathology models. Together, these developments will establish cytoskeletal organization as a controllable, engineerable lever for directing stem cell fate and enabling functional tissue formation.