Engineered presentation of neural cell adhesion molecules for directed neural and neural stem cell behaviors.

摘要

Neurotraumatic injuries result in an irreplaceable cell loss and concomitant deficit in motor and sensory functions. Cell transplantation therapies could potentially address the deficit of neuronal tissues, but remain challenged by limited survival, organization, and integration of transplanted cells. Substrates designed to present specific neurotrophic cues, in precise configurations are candidates for maintaining cell differentiation in vitro and enhancing integration and survival of transplanted cells in vivo. The goal of this dissertation was to design biologically active interfaces based on key developmental neural cell adhesion molecules previously shown to promote neuritogenesis, neuronal differentiation, and survival of neural cells. Specifically, this thesis focuses on modulating the display of protein fragments derived from L1 cell adhesion molecule and N-cadherin and examining cellular responses.;We investigated the efficacy of L1-derived peptide sequences displayed via non-permissive human albumin nanoparticles, which elicited modest neuronal adhesion and neurite outgrowth of primary neurons. In contrast, substrate-bound L1-Fc chimera promoted enhanced neuronal responses. Following this result, we utilized protein A to maximize L1-Fc effectiveness and yield a systematically oriented, multivalent presentation compared to passive adsorption methods. Protein A-presented L1-Fc, displayed from polymeric substrates, greatly improved neurite outgrowth of spinal cord and cerebellar neurons and neuronal differentiation of human embryonic stem cell-derived neural stem cells (hESC-NSCs), compared to L1-Fc presented from the cationic polymer, poly-D-lysine. Next, we sought to address limitations of L1 functionalized substrates, namely, inadequate L1-mediated cell adhesion and limited lineage restriction of hESC-NSCs. To this end, we investigated the effects of presenting N-cadherin-Fc and L1-Fc on differentiation, neurite outgrowth, and survival of hESC-NSCs. Low density N-cadherin substrates promoted greater neuronal differentiation and survival of hESC-NSCs. Enhanced neurite outgrowth and neuronal differentiation was observed in hESC-NSCs cultured on N-cadherin-/L1-Fc substrates, demonstrating the synergistic effect of these two fragments. Findings from this thesis support the paradigm of designing stem cell-bioactive materials by fine-tuning surface concentrations and microscale organization of ligands that regulate different stages of neural development. Such materials could be candidates for recapitulating the microenvironment in the context of biomimetic materials for neural developmental models as well as transplantation devices for neural tissue engineering.