Engineered Biochemical and Biophysical Cell–Matrix Interactions Synergistically Promote Neural Maturation within Viscoelastic Matrices

Introduction: Neural progenitor cells (NPCs) hold immense potential as therapeutic candidates for nervous system regeneration as well as in vitro models of neurodevelopment and disease. However, it is not well understood how NPCs simultaneously incorporate biochemical and biophysical cell–matrix interactions as well as cell–cell communication to regulate differentiation and fate acquisition. In particular, the native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force, yet most conventional biomaterials are formed via static covalent crosslinks and are therefore highly elastic. Furthermore, viscoelastic materials such as brain tissue will undergo stress relaxation, such that when a force is applied, the material can be remodeled to dissipate that force. By incorporating dynamic covalent bonds in a fully defined, hyaluronan–elastin-like protein (HELP) hydrogel, we present the development of a three-dimensional (3D) biomaterial platform with tunable stress relaxation that can be remodeled by cell-imposed forces. To tune biochemical matrix signaling, modularly designed elastin-like proteins (ELPs) were engineered to present one of the following sequences: 1) an N-cadherin mimic sequence (HAVDI) to artificially induce cell–cell signaling, 2) a fibronectin-derived, integrin-binding sequence (RGD) to mimic cell–matrix signaling, or 3) a scrambled, non-integrin-binding sequence (RDG) to which cells cannot bind. Using both murine NPCs and human induced pluripotent stem cell (hiPSC)-derived NPCs, we demonstrate that these hydrogels enable study of cell–cell and cell–matrix interactions, with enhanced neuronal differentiation in biomaterials with faster stress relaxation rates and combined presentation of HAVDI and RGD ligands.


Materials and Methods: ELP is recombinantly expressed and functionalized with hydrazine groups to create ELP-hydrazine. Hyaluronan (HA) is functionalized with propargylamine to obtain HA-alkyne, after which it is modified with either an aldehyde or benzaldehyde to obtain HA-ALD or HA-BZA, respectively. Mechanical characterization of the hydrogels is performed using cone and plate, oscillatory shear rheometry. Stress relaxation measurements are performed with 10% strain. Human NPCs differentiated using previously described dual-SMAD inhibition protocols or adult murine NPCs from micro-dissected dentate gyrus were encapsulated as single cells in the 3D hydrogels. Bulk RNA sequencing was performed on samples immediately post-encapsulation and seven days post-encapsulation. Cell viability, metabolism, gene expression, and protein expression are evaluated at different timepoints post-encapsulation, and we also used immunocytochemistry to observe phenotypic differences in neurite extension.


Results, Conclusions, and Discussions: The hydrogel stiffness is controlled by altering the total number of crosslinks to reach a similar stiffness for all conditions (800-1000 kPa). The stress relaxation rate is controlled by altering the kinetics of crosslink dynamics, by replacing HA-BZA (slow kinetics) with HA-ALD (fast kinetics). Independently, the biochemical properties are customized by altering the amino acid sequence within the ELP. We demonstrate that these dynamically crosslinked HELP hydrogels facilitate gel remodeling and support NPC viability. Furthermore, NPCs encapsulated within these gels underwent relaxation rate dependent neuronal 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 more mature neuronal expression profiles. Conversely, NPCs within static, non-stress-relaxing matrices exhibited limited neurite outgrowth and adopted transcriptomic profiles indicative of intermediate progenitors. By altering the adhesive ligand present (integrin-binding RGD vs. non-integrin-binding RDG), we identified RGD-initiated integrin signaling to be necessary for mechanosensitive neurite outgrowth. Combining HAVDI and RGD ligands further increased neuritic projections in fast-relaxing biomaterials. By inhibiting actin polymerization within fast-relaxing gels, we observed decreased neuritic projections and a concomitant decrease in the expression of neural maturation genes. Through independent manipulation of both external biophysical signaling (i.e., matrix stress relaxation rate) and intracellular cytoskeletal regulation (i.e., inhibition of actin polymerization), we identified the ability to extend neurites as a crucial morphological cue guiding NPC fate acquisition. Taken together, these results suggest that cell-mediated strain within remodelable fast stress-relaxing gels drive neuronal maturation and that tuning biochemical and biomechanical signaling cues within engineered hydrogels has significant potential to advance in vitro models of the neural microenvironment, including potential applications in drug discovery and disease modeling.


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