Matrix viscoelasticity and N-cadherin signaling enhance neural differentiation within engineered 3D hydrogels

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, as they are capable of differentiating into the major lineages of the central nervous system (neurons, astrocytes, and oligodendrocytes). However, it is not well understood how NPCs simultaneously incorporate biochemical and biophysical cell–matrix interactions to regulate NPC stemness and differentiation capacity. 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 elastomeric. 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. We previously demonstrated that degradation-mediated remodeling of 3D hydrogels promoted NPC differentiation and maturation, but degradable matrices undergo irreversible enzymatic degradation, limiting their stability over time. As an alternative materials-based strategy, we incorporate dynamic covalent bonds in a fully defined, hyaluronan–elastin-like protein (HELP) hydrogel, resulting in a three-dimensional (3D) biomaterials platform with tunable stress relaxation that can be remodeled by cell-imposed forces. We then incorporate cell-instructive ligands into the matrix that are present in early developmental stages to further promote the differentiation capacity of NPCs.

Materials and

Methods: To tune biochemical matrix signaling, modularly designed elastin-like proteins (ELPs) are 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. To create viscoelastic, stress-relaxing HELP gels, 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. Additionally, we develop HELP gels with permanent static bonds (norbornene/tetrazine) as an elastic, non-stress-relaxing control. Mechanical characterization of the hydrogels is performed using cone and plate, oscillatory shear rheometry. Stress relaxation measurements are performed with 10% strain. Adult murine NPCs micro-dissected from the dentate gyrus are encapsulated as single cells in the 3D hydrogels. Cell viability, metabolism, gene expression, and protein expression are evaluated at different timepoints post-encapsulation. We use immunocytochemistry to observe phenotypic differences in neurite extension and calcium imaging to functionally validate neuronal differentiation.

Results, Conclusions, and Discussions: We first tuned the mechanical properties of our hydrogels to achieve a range of stiffness and stress relaxation rates. The hydrogel stiffness is controlled by altering the total number of crosslinks to reach a similar stiffness for all conditions (800-1000 kPa) (Fig. 1a). The stress relaxation rate is controlled by altering the kinetics of crosslink dynamics, by replacing HA-BZA (slow kinetics) with HA-ALD (fast kinetics) (Fig. 1b,c). Independently, the biochemical properties are customized by altering the amino acid sequence within the ELP. NPCs cultured in RGD-presenting, viscoelastic gels (both fast- and slow-relaxing) exhibit higher proliferation, metabolic activity, and expression of the NPC markers Nestin and Sox2 compared to NPCs cultured in static, non-stress-relaxing gels. Within these viscoelastic gels, NPCs spontaneously form neurospheres that permit cadherin-mediated cell–cell contact, which we have previously identified as critical to maintaining NPC differentiation capacity. Given the importance of cadherin-mediated signaling, we next hypothesized that viscoelastic HELP gels presenting the cell-instructive ligand HAVDI (an N-cadherin mimic) would enhance neural differentiation and promote neurite outgrowth. Morphological differences emerge as a function of both cell-instructive ligand presentation and matrix stress relaxation rate. Specifically, when both RGD and HAVDI ligands are present in fast-relaxing gels, NPCs no longer cluster as neurospheres and instead exhibit extensive neurite extension throughout the gel while still maintaining expression of NPC markers (Fig. 1d). By independently tuning material properties in our well-defined HELP system, we find that the presence of RGD, the presence of HAVDI, and fast stress relaxation rates are all necessary for uniform cell spreading and neural differentiation. The resulting glial cells express mature glial markers (GFAP and S100β). The resulting neurons exhibit hallmarks of maturity, including expression of mature neuronal markers (MAP2 and βIII-tubulin) and altered calcium signaling in response to neurotransmitter treatment. These results suggest a synergistic effect of N-cadherin signaling and matrix viscoelasticity on regulating NPC differentiation and maturation. Ultimately, engineered hydrogels that incorporate both cell–cell and cell–matrix interactions may increase the regenerative potential of NPC therapies.

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