Stress relaxation timescale and hydrogel network connectivity regulate neural progenitor cell stemness and differentiation

Neural progenitor cells (NPCs) are promising candidates for cell replacement therapies, yet maintaining stemness while enabling expansion in chemically defined three-dimensional (3D) hydrogels remains a challenge. By tuning crosslink exchange kinetics, crosslinker functionality and stoichiometry, polymer phase separation behavior, and adhesive ligand presentation, a family of hydrogels was prepared to study the effects of stress relaxation timescale and network connectivity on NPC phenotype. Hydrogels with rapid relaxation and low connectivity promote expansion of NPCs as distributed single-cell networks that maintain stemness marker expression and differentiation capacity. NPCs embedded in slowly relaxing hydrogels maintained stemness marker expression through cell clustering but exhibited impaired proliferation and differentiation. Similarly, in the absence of integrin-binding cell adhesive ligands, NPCs also maintained stem cell marker expression but remained as clusters rather than distributed single-cell networks. Cadherin cell-cell contacts enable downstream β-catenin signaling and stemness maintenance, which are enhanced in rapidly relaxing, low connectivity networks. These findings identify a combination of network connectivity, stress relaxation timescale, and integrin-binding adhesive ligands as crucial design parameters for maintaining NPC stemness and differentiation capacity in 3D hydrogel networks.