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 growth and alignment of astrocytes and neural progenitor cells and can influence the progenitor cell differentiation. To provide physical guidance, micropatterned polystyrene (PS) substrates were fabricated and chemically modified with poly-L-lysine (PLL) and laminin. Rat postnatal type-1 astrocytes or AHPCs cultured on these substrates align along the grooves of the patterned surface. AHPCs appear highly elongated and often extend their processes in the direction of the grooves and along groove boundaries. To further explore the outgrowth and differentiation of the AHPCs, we have integrated the physical guidance cues along with the biological influence of astrocytes. AHPCs cocultured in contact with astrocytes preferentially acquired neuronal morphology, with nearly double the percentage of cells expressing class III |3-tubulin (TuJl) 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 the AHPCs. Through multi-dimensional cell-cell and cell-matrix interactions, 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 this environment, the aligned astrocytes may present discrete guidance cues to the overlying AHPCs involving contact mediated or soluble factors or a combination of both. Using a non-contact co-culture system, it was determined that astrocyte-derived soluble factors can enhance neurite outgrowth and induce neuronal differentiation of the AHPCs with significantly more cells immunoreactive for TuJl in the noncontact co-culture system than in the contact co-culture system. Therefore, soluble cues may have had a stronger influence on neuronal differentiation and neuritic extension compared to the contact mediated factors or the 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 studies as opposed to the noncontact co-cultures. This research provides insights into mechanisms of neural stem cell differentiation as well as a foundation for the development of a promising nerve regeneration strategy that incorporates a synergistic combination of cues for guided central nervous system repair following