Vitronectin establishes a differentiation-restrictive extracellular microenvironment that sustains myoblast proliferation across species.

Abstract

The extracellular matrix (ECM) critically regulates cell fate decisions, yet matrix components that actively impose differentiation-restrictive states remain insufficiently defined from a biomaterials perspective. Here, we identify vitronectin (VN), a major serum-derived ECM glycoprotein, as a differentiation-restrictive matrix component that stabilizes a proliferation-competent microenvironment for myoblast expansion. Substrate-associated VN markedly suppresses myogenic differentiation of C2C12 cells, as demonstrated by reduced myotube formation and attenuated expression of canonical differentiation markers. Mechanistically, this effect is mediated through integrin αvβ3–dependent signaling, highlighting a defined matrix-dependent pathway by which matrix engagement modulates lineage progression. In parallel, VN sustains proliferative activity under differentiation conditions by maintaining mitotic progression, preserving growth factor receptor phosphorylation, and preventing cell cycle exit. Importantly, VN-dependent regulation is preserved in three-dimensional spheroid culture and in primary embryonic chicken myogenic cells, demonstrating context-independent matrix functionality. Furthermore, defined serum-free culture experiments reveal that VN, in cooperation with leukemia inhibitory factor (LIF), establishes a controllable expansion platform that maintains reversible differentiation competence. Together, these findings define vitronectin as a matrix element that enforces a differentiation-restrictive microenvironment and provide design principles for engineering ECM-based systems to regulate progenitor cell expansion.