Biomimetic Polymer Scaffolds for Neural Stem Cell Adhesion & Differentiation

Neurodegenerative diseases and neurotraumatic injuries result in an irreplaceable cell loss and concomitant deficit in motor and sensory functions.  An ideal strategy for repair will allow for cell survival, migration, and integration of exogenous cells with host tissue. Biomaterials provide a potential strategy to elicit these responses by presentation of specific cues to control cellular behavior.  While much of current biomaterials research has focused on optimizing the mechanical and soluble cues required to direct neural cell behavior, adhesive cues and, more importantly, cell-cell interactions may also play a role.  In this study, we investigated the role of differential presentation of fragments of L1, a transmembrane, homophilic binding, neural cell adhesion molecule, as a biointerfacial strategy to direct neuronal cell retention and differentiation of neural stem cells on implantable scaffolds, while inhibiting the adhesion of non-neuronal phenotypes, such as astrocytes and fibroblasts.  This approach is hypothesized to not only promote the survival and outgrowth phenomena of neuronally developed cells, but also guide the development of neural stem cells as L1 has been implicated in the normal development of the central nervous system.

Two-dimensional films of poly(desaminotyrosyl tyrosine ethyl ester carbonate) (poly(DTE carbonate) polymers were used as the biomaterial model for L1 functionalization as these polymers are highly biocompatible and can be tuned to alter protein adsorption, hydration/biomechanical properties, and degradation behaviors.  Various configurations of L1 presentation were compared in terms of relative concentrations, orientations, and multivalency of L1 display, including passive adsorption on cationic, poly-D-lysine-treated substrates or L1-Fc fused fragment presentation from protein A-coated polymer substrates.  Protein A-based presentation of L1-Fc was hypothesized to result in the outward display of L1 while resulting in a clustered ligand presentation of L1 and increase ligand efficacy, as well as enhance cell binding affinity and L1-mediated intracellular signaling.

We report that the configuration of L1 presentation marked affected neural cell behavior on the biofunctionalized polymer substrates.  When presented through an in vivo-like configuration on protein A, L1 significantly increased neurite outgrowth of spinal cord neurons compared to the traditional presentation of passive adsorption on poly-D-lysine as early as 24 hours.  After 72 hours, while both conditions supported the formation of neural networks, protein A-presented L1 resulted in a denser population of extended neurites.  Next, the role of L1 on active differentiation of human neural stem cells was examined.  Neural stem cells derived from H9 human embryonic stem cells were used for these studies.  Protein A presented-L1 yielded quantitative enhancement in levels of neuronal differentiation and outgrowth of human neural stem cells in comparison to PDL-presented L1 after 7 days in culture.  This approach is being integrated within three-dimensional scaffolds for the design of neural stem cell-transplantable constructs for the management of spinal cord injury.