Vitronectin defines a differentiation-restraining extracellular matrix state in myogenic cells

Skeletal muscle differentiation is classically triggered by reducing serum-derived mitogenic cues; however, the extracellular matrix-associated factors that actively maintain a proliferative, differentiation-resistant state remain poorly defined. Here, we identify vitronectin (VN) as a key extracellular matrix gatekeeper that controls myogenic cell fate. VN is abundant in fetal bovine serum but largely absent from horse serum, rapidly declines upon differentiation induction, and selectively suppresses myogenic differentiation while sustaining proliferation through integrin v{beta}3-dependent signaling. VN maintains growth factor receptor activity and cell-cycle progression even under differentiation conditions, thereby preventing myoblast commitment. These effects extend to three-dimensional culture and primary embryonic chicken myogenic cells, indicating evolutionary conservation. Importantly, VN progressively accumulates during long-term passaging, coinciding with replicative senescence and impaired differentiation, and inhibition of v{beta}3 signaling partially restores myogenic competence in senescent cells. Together, our findings redefine a long-standing myoblast culture paradigm by identifying a serum-derived extracellular matrix protein as a decisive regulator linking extracellular environment, proliferative niche maintenance, and age-associated decline in myogenic competence.