Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity

Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.