Directed growth and selective differentiation of neural progenitor cells using a synergistic combination of topographical and soluble cues

The purpose of this study is to develop strategies to facilitate nerve regeneration using a synergistic combination of guidance cues. We are investigating the cellular mechanisms of development using adult rat hippocampal progenitor cells (AHPCs) and have demonstrated that manipulating a combination of physical, chemical and biological cues can lead to oriented astrocyte and neural progenitor cell outgrowth and can influence progenitor cell differentiation. To provide physical guidance, micropatterned polystyrene (PS) substrates were fabricated and chemically modified with laminin. Astrocytes or AHPCs cultured on these substrates align along the grooves of the patterned surface extending highly elongated processes. To further explore AHPC outgrowth and differentiation, physical guidance cues were integrated with the biological influence of astrocytes. AHPCs co-cultured in contact with astrocytes preferentially acquired neuronal morphology, with nearly double the percentage of cells expressing class III beta-tubulin (TuJ1) on the micropatterned half of the substrate, as opposed to the planar half of the substrate, or compared to those growing in the absence of astrocytes. This indicates that substrate three-dimensional topography, in synergy with chemical (laminin) and biological (astrocytes) guidance cues, facilitates neuronal differentiation of AHPCs. This environment provided biological and spatial control over differentiation enhancing neuronal differentiation and promoting neurite alignment on topographically different regions of the same substrate. In a non-contact co-culture system, astrocyte-derived soluble factors enhanced neurite outgrowth and induced neuronal differentiation with significantly more AHPCs TuJ1 immunoreactive than in the contact co-culture. Therefore, soluble cues may have had a stronger influence on neuronal differentiation and neuritic extension compared to contact mediated factors or a combination of soluble and contact mediated factors that were presented by the monolayer of aligned astrocytes. The results also point to the potential role of localized concentration of these factors within the microgrooves as a reason for the differences in differentiation on micropatterned and planar substrates in the contact as opposed to the non-contact co-cultures. This research provides insights into mechanisms of neural stem cell differentiation and a foundation for the development of a promising nerve regeneration strategy incorporating a synergistic combination of cues for guided central nervous system repair following injury

Micropatterned polymer films for optic nerve regeneration

In an effort to promote optic nerve regeneration in vivo, directional growth of astrocytes has been achieved on polymer substrates in vitro. The purpose of this project was to investigate the cellular mechanisms of optic nerve repair using astrocyte cultures. Manipulating a combination of physical and chemical cues, astrocyte adhesion and alignment in vitro were examined. To provide physical guidance, micropatterned polymer films of polystyrene (PS) were fabricated. Laminin was selectively adsorbed into the grooves of the patterned surface. Rat type-1 astrocytes were seeded onto micropatterned PS substrates of 10 [mu]m groove width, 10 or 20 [mu]m groove spacing and 3 or 4 [mu]m groove depth. The effects of substrate topography and the adsorption of laminin to the PS substrates on the behavior and morphology of the astrocytes were explored. The astrocytes were found to align parallel to the micropatterned grooves at initial seeding densities of approximately 7500, 13,000, and 20,000 cells per cm2 due to the effects of the physical and chemical guidance mechanisms. Adsorbing laminin in the microgrooves of the micropatterned PS substrates improved cell adhesion and spreading of cytoskeletal filaments significantly. At these initial seeding densities, over 85% alignment in the direction of the grooves was achieved on the micropatterned PS substrates with laminin adsorbed in the grooves. The effects of physical and chemical guidance mechanisms on the behavior and morphology of the astrocytes on the PS substrate were explored to determine their influence on the outgrowth and differentiation of adult neural stem cell cultures in vitro. This combination of guidance cues has the potential to provide a permissive substrate for in vivo regeneration of the diseased or injured optic nerve and other regions of the central nervous system