Effect Of SCF On The Migration Of Differenting NSCs

Malignant gliomas remain virtually incurable because of its highly invasive nature, despite extensive surgical excision, radiation and chemotherapy. In vitro as well as in vivo studies demonstrate that neural stem cells (NSCs) display a strong tendency to migrate toward gliomas and surround invading tumor cells, thus highlighting the potential use of NSCs as delivery vehicles for therapeutic genes. However, the mechanisms and factors that regulate migration are not well understood. Gliomas release numerous chemokines and growth factors that are capable of stimulating the directed migration of exogenous and endogenous NSCs into the tumor microenvironment. Here, we focus on the effect of stem cell factor (SCF), one of the factors that are present at the injury sites, on the migration of differentiating NSCs.In this study, we isolated and purified NSCs from the subventricular zone (SVZ) of newborn SD rats and induced these cells to differentiate with 1% fatal calf serum (FCS). These NSCs were characterized by morphology, antigen expression (nestin, GFAP andβ-III-tubulin) and differentiation potential and results showed that the majority of cells are immunopositive for nestin, indicating that they are undifferentiated neural progenitors. After the withdrawal of FGF-2, cells differentiate into GFAP containing astrocytes,β-III-tubulin-positive neurons.NSCs from SVZ were cultured for 4 days or 5 days, and then changed to the medium with 1% FCS for differentiation. SCF at 5 ng/ml or 25 ng/ml was added to the differentiating cells. The migratory capability of NSCs at different differentiation states was analyzed by time-lapse video analysis. Our results indicated that treatment with SCF at 5 ng/ml didn't influence the migration distance during the differentiation period of 0~12 h and 36~48 h, but significantly increased it in the phase of the differentiation of 12~36 h. In response to 25 ng/ml of SCF, the migration distance of the differentiating NSCs increased significantly in 0-36 h rather than the later period (36-48 h). In contrast, the forward migration index (FMI) of NSCs was improved by 25 ng/ml SCF through the whole differentiation period. These results suggest a close relationship between the migration behavior and the differentiation states of NSCs.Then we investigated SCF-induced migration of C17.2 cells using Boyden chamber and Dunn chamber. Results of Boyden Chamber showed that cells of 24-h differentiation displayed the strongest tropism toward SCF compared with undifferentiated C17.2 cells. After 24 h of differentiation, the migratory responsiveness decreased over time. On the 7th day, less C17.2 cells migrated to SCF than did normal cells that were at an undifferentiated state. The Dunn Chamber results indicated both the migration speed and the forward migration index (FMI) of cells exposed to SCF were significantly greater than those of cells exposed to a uniform concentration of SCF. Cells displayed a directed migratory behavior towards the source of SCF, while they migrated randomly under conditions of uniform SCF distribution. The addition of LY294002, a specific inhibitor of PI3K, significantly altered the pattern of migration of C17.2 cells in response to SCF gradient. The migration speed was significantly decreased compared with untreated cells, while cells remained their directional response to the SCF gradient. These results illustrate that SCF induces the directional migration of C17.2 cells via the increase in the migration efficiency and the migration speed of these cells through activation of PI3K/Akt signaling pathway.Rac1 is a molecular switch that has an inactive GDP-bound and an active GTP-bound state. After activation, Rac1 can participate in the formation of actin stress and adhesion plaque, promote the reform of cytoskeleton, regulate the extension of lamellipodia and filopodia, influence the polarization of cell, and enhance cell migration. p21-activated kinases (PAKs) are the downstream effector proteins of Rho GTPase. Binding of Rac-GTP leads to PAK autophosphorylation and activation of the ability to phosphorylate exogenous substrates on serine and/or threonine residues. Rac activates PAK through binding to the p21-binding domain (PBD). The gene fragment of PBD was amplified by RT-PCR and cloned into the pGEX-4T-1 prokaryotic expression vector, DNA sequencing confirmed that the expression vector of pGEX-4T-1/ PBD was constructed successfully and fusion protein was expressed effectively in Escherichia coli, with about 95 % of the GST-PBD fusion protein. This study lays the basis for the research on the relationship between the Rac1 activity and the differentiation states of NSCs.Taken together, these results illustrate that SCF induces the directional migration of NSCs via the increase in the migration efficiency and the migration speed of these cells through activation of PI3K/Akt, contributing to better understanding of the migration and differentiation mechanisms of NSCs, and thus helping design the therapeutic strategies to treat central nervous system (CNS) diseases.