The purpose of this study was to investigate the ability of astrocyte-derived factors to influence neural progenitor cell differentiation. We previously demonstrated that rat adult hippocampal progenitor cells (AHPCs) immunoreactive for the neuronal marker, class III β-tubulin (TUJ1) were significantly increased in the presence of astrocyte-derived soluble factors under non-contact co-culture conditions. Using whole cell patch clamp analysis, we observed that the co-cultured AHPCs displayed two prominent voltage-gated conductances - tetraethyl ammonium (TEA)-sensitive outward currents and fast transient inward currents. The outward and inward current densities of the co-cultured AHPCs were approximately 2.5-fold and 1.7-fold greater, respectively, than those of cells cultured alone. These results suggest that astrocyte-derived soluble factors induce neuronal commitment of AHPCs. To further investigate the activity of a candidate neurogenic factor on AHPC differentiation, we cultured AHPCs in the presence or absence of purified rat recombinant interleukin-6 (IL-6). We also confirmed that the astrocytes used in this study produced IL-6 by ELISA and RT-qPCR. When AHPCs were cultured with IL-6 for 6-7 days, the TUJ1-immunoreactive AHPCs and the average length of TUJ1-immunoreactive neurites were significantly increased, compared to the cells cultured without IL-6. Moreover, IL-6 increased the inward current density to a comparable extent as did co-culture with astrocytes, with no significant differences in the outward current density, apparent resting potential, or cell capacitance. These results suggest that astrocyte-derived IL-6 may facilitate AHPC neuronal differentiation. Our findings have important implications for understanding injury-induced neurogenesis and developing cell-based therapeutic strategies using neural progenitors.

Adult rat hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitor cells (NPCs) that can differentiate into neurons, oligodendrocytes, and astrocytes. AHPCs contact a variety of molecular cues within their surrounding microenvironment via integrins. We hypothesize that integrin receptors are important for NPCs. In this study, we have examined the distribution of integrins in neuronal-like, oligodendrocyte-like, and astrocyte-like AHPCs when grown on substrates that support integrin-mediated adhesion (laminin, fibronectin), and those that do not (poly-L: -ornithine, PLO) using immunocytochemistry as well as characterized the phenotypic differentiation of AHPCs plated on laminin and fibronectin. Focal adhesions were prominent in AHPCs plated on purified substrates, but were also found in AHPCs plated on PLO. The focal adhesions observed in AHPCs plated on PLO substrates may be formed by self-adhesion to the endogenously produced laminin or fibronectin. We have demonstrated that integrins contribute to the initial morphological differentiation of AHPCs, as inhibition of fibronectin binding with the competitive inhibitor echistatin significantly decreased the number of processes and microspikes present in treated cells, and also decreased overall cell area. Finally, we have characterized the genetic profile of a subset of integrins and integrin-related genes in the AHPCs using reverse transcriptase polymerase chain reaction. These results demonstrate an important role of integrins, in vitro, for the initial morphological differentiation of AHPCs.

In this study we investigated the differentiation of human neural progenitor cells (hNPCs) in vitro to evaluate their differentiation potential and in vivo to explore their viability and behavior following transplantation. Progenitors were maintained as neurospheres in media containing basic fibroblast growth factor and epidermal growth factor. Micropatterned polystyrene substrates were fabricated and coated with ECL (entactin, collagen, and laminin) to provide physical and chemical guidance during the differentiation of the hNPCs. The hNPCs growing on the micropatterned substrates showed no differences in proliferation or differentiation potential compared with those hNPCs growing on the nonpatterned substrates. However, hNPCs cultured on the micropatterned substrates were aligned in the direction of the micropattern compared with those cells growing on the nonpatterned substrates. Furthermore, hNPC migration was directed in alignment with the micropatterned substrates. Transplantation of the hNPCs into the developing retina was used to evaluate their behavior in vivo. Cells displayed extensive survival, differentiation, and morphological integration following xenotransplant into the retina, even in the absence of immunosuppression. Taken together, our results show that these multipotent hNPCs are a neurogenic progenitor population that can be maintained in culture for extended periods. Although the micropatterned substrates have no major effect on the proliferation or differentiation of the hNPCs, they clearly promoted alignment and directed neurite outgrowth along the pattern as well as directing migration of the cells. These approaches may provide important strategies to guide the growth and differentiation of NPCs in vitro and in vivo.