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Identification of small molecules that direct neural stem cells (NSCs) into specific cell types would be helpful to understand the molecular mechanisms involved in regulation of NSC fate, and facilitate the development of therapeutic applications. In the current study, we developed and screened small molecules that can modulate the fate of NSCs that are derived from rat fetal cortex. Among these compounds, compounds 5 and 6 successfully differentiated NSCs into astrocytes and neurons, respectively. Compound 5 induced astrocytogenesis by increasing expression of interleukin-6, bone morphogenetic protein 2 and leukemia inhibitory factor and through consequent phosphorylation of signal transducer and activator of transcription 3 and Sma- and Mad-related protein 1/5/8 in NSCs. In addition, compound 5 increased the expression of fibroblast growth factor (FGF) 2 and FGF8 which may regulate the branching and morphology of astrocytes. Taken together, our results suggest that these small molecules can serve as a useful tool to study cell fate determination in NSCs and be used as an inexpensive alternative to cytokines to study mechanisms of astrocytogenesis.

P7C3 and its derivatives, 1-(3,6-dibromo-9H-carbazol-9-yl)-3-(p-tolylamino)propan-2-ol (1) and N-(3-(3,6-dibromo-9H-carbazol-9-yl)-2-hydroxypropyl)-N-(3-methoxyphenyl)-4-methylbenzenesulfonamide (2), were previously reported to increase neurogenesis in rat neural stem cells (NSCs). Although P7C3 is known to increase neurogenesis by protecting newborn neurons, it is not known whether its derivatives also have protective effects to increase neurogenesis. In the current study, we examined how 1 induces neurogenesis. The treatment of 1 in NSCs increased numbers of cells in the absence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), while not affecting those in the presence of growth factors. Compound 1 did not induce astrocytogenesis during NSC differentiation. 5-Bromo-2′-deoxyuridine (BrdU) pulsing experiments showed that 1 significantly enhanced BrdU-positive neurons. Taken together, our data suggest that 1 promotes neurogenesis by the induction of final cell division during NSC differentiation.

Recently, the importance of surface nanotopography in the determination of stem cell fate and behavior has been revealed. In the current study, we generated polystyrene cell-culture dishes with an omnidirectional nanopore arrayed surface (ONAS) (diameter: 200 nm, depth: 500 nm, center-to-center distance: 500 nm) and investigated the effects of nanotopography on rat neural stem cells (NSCs). NSCs cultured on ONAS proliferated better than those on the flat surface when cell density was low and showed less spontaneous differentiation during proliferation in the presence of mitogens. Interestingly, NSCs cultured on ONAS at clonal density demonstrated a propensity to generate neurospheres, whereas those on the flat surface migrated out, proliferated as individuals, and spread out to attach to the surface. However, the differential patterns of proliferation were cell density-dependent since the distinct phenomena were lost when cell density was increased. ONAS modulated cytoskeletal reorganization and inhibited formation of focal adhesion, which is generally observed in NSCs grown on flat surfaces. ONAS appeared to reinforce NSC-NSC interaction, restricted individual cell migration and prohibited NSC attachment to the nanopore surface. These data demonstrate that ONAS maintains NSCs as undifferentiated while retaining multipotency and is a better topography for culturing low density NSCs.

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