Matrix Viscoelasticity Regulates the Stemness and Multilineage differentiation of Primary Neural Progenitor-Stem Cells in 3D

Neural progenitor-stem cells (NPSCs) reside in mechanically dynamic brain microenvironments and give rise to neurons, astrocytes, and oligodendrocytes. Although recent studies have shown that matrix viscoelasticity can influence neural maturation and neurogenic differentiation, its role in primary NPSCs beyond neurogenesis remains less defined. How matrix viscoelasticity or stress relaxation regulates primary NPSC stemness, neuronal and glial differentiation, and the associated matrix-cell mechanotransduction pathways are not well understood. Here, we use alginate hydrogels with independently tunable stiffness and stress relaxation properties to investigate how matrix stress relaxation regulates the fate of primary subventricular zone (SVZ)-derived NPSCs in 3D. The results suggested that matrices with faster stress relaxation enhances stemness maintenance, radial glial-like marker expression, and differentiation of NPSCs toward neuronal, astrocytic, and oligodendrocytic lineages in the corresponding biochemical environments. In mixed neuronal/astrocytic differentiation conditions, fast-relaxing matrices preferentially promote neuronal differentiation. Mechanistically, NPSC responses to matrix stress relaxation involve integrin-mediated adhesion, actomyosin contractility, actin polymerization, and Piezo1 activity, with distinct contributions across differentiation lineages. Together, these findings reveal how matrix stress relaxation regulates primary NPSC stemness and multilineage differentiation through multiple mechanotransduction pathways in 3D.