Fibroblasts impair muscle stem cell self-renewal via excessive fibronectin deposition in viscoelastic hydrogel co-cultures

Muscle satellite cells (SCs) regenerate skeletal muscle, but their regenerative capacity declines with age, in part due to extracellular matrix (ECM) remodeling and aberrant fibroblast activation within the SC niche. In regenerating young mouse muscle, fibronectin remodeling is transient, whereas in aged mouse muscle, fibronectin remodeling is prolonged and disorganized. Fibroblasts in aged mice are activated, increasing fibronectin deposition and expressing elevated α-smooth muscle actin (αSMA), which negatively influence SC fate. We develop a viscoelastic hydrogel co-encapsulation system, enabling three-dimensional co-culture of intact myofibers with primary fibroblasts. Using this 3D co-culture system, we show that fibroblasts from young mice support SC quiescence and self-renewal, whereas fibroblasts from aged mice aberrantly activate SCs and promote their differentiation on myofibers isolated from either young or aged mice. Knocking down fibronectin ( Fn1 ) in fibroblasts from aged mice partially restores SC function, promoting quiescence and limiting differentiation. Using a novel 3D hydrogel co-culture system, we demonstrate that fibroblast-deposited fibronectin is a key age-associated regulator negatively affecting SC fate within the SC niche of aged mice.