Engineered N-cadherin and L1 biomimetic substrates concertedly promote neuronal differentiation, neurite extension and neuroprotection of human neural stem cells.

We investigated the design of neurotrophic biomaterial constructs for human neural stem cells, guided by neural developmental cues of N-cadherin and L1 adhesion molecules. Polymer substrates fabricated either as two-dimensional (2-D) films or three-dimensional (3-D) microfibrous scaffolds were functionalized with fusion chimeras of N-cadherin-Fc alone and in combination with L1-Fc, and the effects on differentiation, neurite extension and survival of H9 human-embryonic-stem-cell-derived neural stem cells (H9-NSCs) were quantified. Combinations of N-cadherin and L1-Fc co-operatively enhanced neuronal differentiation profiles, indicating the critical nature of the two complementary developmental cues. Notably, substrates presenting low levels of N-cadherin-Fc concentrations, combined with proportionately higher L1-Fc concentration, most enhanced neurite outgrowth and the degree of MAP2+ and neurofilament-M+ H9-NSCs. Low N-cadherin-Fc alone promoted improved cell survival following oxidative stress, compared to higher concentrations of N-cadherin-Fc alone or combinations with L1-Fc. Pharmacological and antibody blockage studies revealed that substrates presenting low levels of N-cadherin are functionally competent so long as they elicit a threshold signal mediated by homophilic N-cadherin and fibroblast growth factor signaling. Overall, these studies highlight the ability of optimal combinations of N-cadherin and L1 to recapitulate a "neurotrophic" microenvironment that enhances human neural stem cell differentiation and neurite outgrowth. Additionally, 3-D fibrous scaffolds presenting low N-cadherin-Fc further enhanced the survival of H9-NSCs compared to equivalent 2-D films. This indicates that similar biofunctionalization approaches based on N-cadherin and L1 can be translated to 3-D "transplantable" scaffolds with enhanced neurotrophic behaviors. Thus, the insights from this study have fundamental and translational impacts for neural-stem-cell-based regenerative medicine.

Oriented, multimeric biointerfaces of the L1 cell adhesion molecule: an approach to enhance neuronal and neural stem cell functions on 2-D and 3-D polymer substrates.

This article focuses on elucidating the key presentation features of neurotrophic ligands at polymer interfaces. Different biointerfacial configurations of the human neural cell adhesion molecule L1 were established on two-dimensional films and three-dimensional fibrous scaffolds of synthetic tyrosine-derived polycarbonate polymers and probed for surface concentrations, microscale organization, and effects on cultured primary neurons and neural stem cells. Underlying polymer substrates were modified with varying combinations of protein A and poly-D-lysine to modulate the immobilization and presentation of the Fc fusion fragment of the extracellular domain of L1 (L1-Fc). When presented as an oriented and multimeric configuration from protein A-pretreated polymers, L1-Fc significantly increased neurite outgrowth of rodent spinal cord neurons and cerebellar neurons as early as 24 h compared to the traditional presentation via adsorption onto surfaces treated with poly-D-lysine. Cultures of human neural progenitor cells screened on the L1-Fc/polymer biointerfaces showed significantly enhanced neuronal differentiation and neuritogenesis on all protein A oriented substrates. Notably, the highest degree of ßIII-tubulin expression for cells in 3-D fibrous scaffolds were observed in protein A oriented substrates with PDL pretreatment, suggesting combined effects of cell attachment to polycationic charged substrates with subcellular topography along with L1-mediated adhesion mediating neuronal differentiation. Together, these findings highlight the promise of displays of multimeric neural adhesion ligands via biointerfacially engineered substrates to "cooperatively" enhance neuronal phenotypes on polymers of relevance to tissue engineering.

E-cadherin-expressing feeder cells promote neural lineage restriction of human embryonic stem cells.

Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article, we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule, E-cadherin, to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin, but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase, suggesting that E-cadherin promotes rapid neuronal differentiation. Further, these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin, neurofilament, and neural cell adhesion molecule. Moreover, blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation, we are able to utilize a specific cell-cell adhesion molecule, E-cadherin, to influence the nature and degree of neural specialization.