Soluble factors from neocortical astrocytes enhance neuronal differentiation of neural progenitor cells from adult rat hippocampus on micropatterned polymer substrates.

Rat adult hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitors that have the ability to differentiate into neurons and glia. Previously, we demonstrated that coculture of AHPCs with postnatal day 2, type 1 cortical astrocytes on laminin-coated micropatterned polymer substrates facilitates selective neuronal differentiation of the AHPCs (Recknor et al., Biomaterials 2006;27:4098-4108). Under this condition, multidimensional cell-cell and/or cell-extracellular matrix interactions, as well as possible soluble factors released from astrocytes provided spatial and temporal control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. To investigate the potential role of astrocyte-derived soluble factors as cues involved in neuronal differentiation, a noncontact coculture system was used. Under control conditions, approximately 14% of the AHPCs were immunoreactive (IR) for the neuronal marker, class III beta-tubulin (TUJ1-IR). When cocultured in physical contact with astrocytes, neuronal differentiation increased significantly to about 25%, consistent with our previous results. Moreover, under noncontact coculture conditions using Transwell insert cultures, neuronal differentiation was dramatically increased to approximately 64%. Furthermore, neurite outgrowth from neuronal cell bodies was considerably greater on the patterned substrate when compared with the nonpatterned planar substrate under noncontact coculture conditions. Taken together, our results demonstrate that astrocyte-derived soluble factors provide cues for specific neuronal differentiation of AHPCs cultured on micropatterned substrates. In addition, a suppressive influence on neuronal differentiation appears to be mediated by contact with cocultured astrocytes. These results provide important insights into mechanisms for controlling neural progenitor/stem cell differentiation and facilitate development of strategies for CNS repair.

Directed growth and selective differentiation of neural progenitor cells on micropatterned polymer substrates.

Directional growth and differentiation of adult rat hippocampal progenitor cells (AHPCs) were investigated on micropatterned polymer substrates in vitro. Astrocytes or AHPCs cultured on micropatterned polystyrene substrates chemically modified with laminin exhibited over 75% alignment in the groove direction. AHPCs co-cultured with astrocytes preferentially acquired neuronal morphology, with nearly double the percentage of cells expressing class III beta-tubulin 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 interactions, this environment provides spatial control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. Integrating these cues is important in understanding and controlling neural stem cell differentiation and designing scaffolds for guided nerve regeneration.

Growth and differentiation of astrocytes and neural progenitor cells on micropatterned polymer films.

This paper investigates the influence of micropatterned polymers and chemical modification on neural progenitor cell growth and differentiation in co-culture systems with astrocytes. We sought to develop strategies to facilitate nerve regeneration using a synergistic combination of guidance cues, investigating the cellular mechanisms of nerve repair using adult rat hippocampal progenitor cells (AHPCs). Our studies have shown that this synergistic combination of physical, chemical, and biological cues can lead to oriented growth of astrocytes and progenitor cells, can control and accelerate neurite outgrowth and alignment in vitro, and may influence differentiation of progenitor cells.