5-Azacitidine Partially Resets the Subcellular Localization of YAP in Human Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) sense biophysical cues from their microenvironment, which regulate cytoskeletal organization and the nuclear–cytoplasmic distribution of the mechanotransducer Yes-associated protein (YAP), thereby shaping cellular behavior. Prolonged ex vivo culture on non-physiologically rigid substrates induces persistent nuclear YAP localization, a phenomenon often referred to as mechanical memory. We therefore examined whether transient epigenetic modulation could modulate YAP subcellular localization in human bone marrow-derived MSCs. Treatment with the DNA methyltransferase inhibitor 5-azacitidine (5-Aza) shifted YAP localization toward the cytoplasm in MSCs, without overt changes in pluripotency marker expression or neural differentiation capacity. RNA sequencing revealed broad down-regulation of extracellular matrix (ECM)-related genes following 5-Aza treatment. Independent suppression of ECM production via TGF-β signaling similarly promoted cytoplasmic YAP localization. When subsequently transferred to soft substrates, 5-Aza–treated MSCs restored YAP relocalization despite prior expansion on stiff surfaces. Together, these findings suggest that transient 5-Aza treatment can partially alleviate mechanically induced YAP regulation associated with mechanical memory. Thus, simple and transient administration of 5-Aza may offer a practical means to improve the quality of MSCs during ex vivo expansion for cell-based therapies.