Nuclear compression overrides matrix softness to regulate stem cell fate through actomyosin-driven YAP mechanotransduction

Mechanical cues play a pivotal role in regulating stem cell fate, yet how cells integrate compressive forces in compliant microenvironments remains poorly understood. Here, we show that nuclear compression overrides soft matrix cues to induce osteogenic differentiation in mesenchymal stem cells through an Integrin/vinculin-independent, cytoskeleton-mediated mechanism. Using a microfabricated polydimethylsiloxane (PDMS) confiner, we applied defined vertical confinement (3 and 10 μm) to mesenchymal stem cells (MSCs) cultured on substrates of varying stiffness. On soft substrates, compression triggered a marked increase in cell spreading, actomyosin contractility and cortical stiffness, leading to YAP nuclear translocation and upregulation of osteogenic transcription factors such as RUNX2. Surprisingly, this response occurred in the absence of canonical integrin-vinculin signaling, but was abolished by pharmacological inhibition of actin polymerization or Myosin II activity. Prolonged intermittent confinement promoted robust osteogenesis on soft substrates, as demonstrated by Alkaline Phosphatase (ALP) staining and gene expression, and was suppressed by YAP inhibition. Our findings uncover a previously unrecognized nuclear mechanosensing axis, in which cells actively generate internal tension to counteract external compression and engage YAP-mediated transcriptional programs, providing a tunable strategy for directing stem cell fate in soft environments. STATEMENT OF SIGNIFICANCE: Mechanical control of stem cell fate is traditionally attributed to extracellular matrix stiffness. Here, we show that vertical nuclear confinement functions as a dominant mechanical cue that can override soft matrix signals to direct cell differentiation. Using a precisely controlled confinement system, we find that confinement induces a cytoskeletal reinforcement response that occurs largely independent of canonical integrin-vinculin adhesion signaling, leading to increased actomyosin contractility, cortical stiffening, and YAP nuclear localization. This tension-driven nuclear mechanosensing enables robust osteogenic commitment even in otherwise non-permissive soft environments. Our findings reveal a distinct mode of mechanotransduction in which cells elevate intracellular tension to counter external confinement, highlighting nuclear confinement as a key regulator of stem cell fate in confined microenvironments.