Tunable Hydrogel Viscoelasticity Modulates Human Neural Maturation

Human induced pluripotent stem cells (hiPSCs) have emerged as a promising in vitro model system for studying neurodevelopment. However, current models remain limited in their ability to incorporate tunable biochemical and biomechanical signaling cues imparted by the neural extracellular matrix (ECM). The native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force. To recapitulate the remodelability of the neural ECM, we developed a family of protein-engineered hydrogels crosslinked with either static or dynamic covalent bonds that exhibit tunable stress relaxation rates. hiPSC-derived neural progenitor cells (NPCs) encapsulated within these gels underwent relaxation rate-dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections, exhibited decreased metabolic activity, and expressed higher levels of genes associated with neural maturation. By inhibiting actin polymerization, we observed decreased neuritic projections and a concomitant decrease in the expression of neural maturation genes. Taken together, these results suggest that microenvironmental viscoelasticity is sufficient to bias human NPC maturation. Overall design: To explore the effects of matrix viscoelasticity on hiPSC differentiation and maturation, we encapsulated hiPSC-derived NPCs in protein engineered matrices with three distinct stress relaxation profiles and collected cells at cells after seven days.

人类诱导多能干细胞（human induced pluripotent stem cells，hiPSCs）已成为研究神经发育的极具潜力的体外模型体系。然而，当前模型在整合神经细胞外基质（neural extracellular matrix，ECM）所提供的可调控生化与生物力学信号方面仍存在局限。天然脑源性细胞外基质具有粘弹性与应力松弛特性，对所施加的外力呈现时间依赖性响应。为重现神经细胞外基质的可重塑特性，我们开发了一类由静态或动态共价键交联的蛋白质工程水凝胶，其应力松弛速率可调控。封装于此类凝胶中的hiPSC源性神经前体细胞（neural progenitor cells，NPCs）会呈现出依赖于应力松弛速率的成熟过程。具体而言，应力松弛速率更快的水凝胶内的神经前体细胞可伸出更长且更复杂的神经突起，代谢活性降低，且表达更高水平的神经成熟相关基因。通过抑制肌动蛋白聚合，我们观察到神经突起生成减少，且神经成熟基因的表达也随之降低。综上，本研究结果表明，微环境粘弹性足以调控人类神经前体细胞的成熟过程。总体实验设计：为探究基质粘弹性对hiPSC分化与成熟的影响，我们将hiPSC源性神经前体细胞封装于具有三种不同应力松弛特征的蛋白质工程基质中，并于7天后收集细胞。