Differentiation Of Neural Stem Cells Influences Their Chemotactic Responses To VEGF

A precise migration of neural stem/progenitor cells (NSCs) is prerequisite during development for the formation of the nervous system and plays a pivotal role in a variety of both physiological and pathological events in adult. Many neurodegenerative diseases are closely related to the deficient migration of NSCs. Increasing studies have demonstrated that NSCs, either endogenous or transplanted, are highly motile and display a unique tropism for areas of pathology in the adult brain. This phenomenon reveals a therapeutic potential of NSCs for the neural regeneration and nerve repair after injury.There is much yet to learn, however, about the mechanisms that regulate the directed migration of NSCs. Many factors, including vascular endothelial growth factor (VEGF), an angiogenic factor critical in vasculargeneis and angiogenesis, are involved in the navigation of NSCs to their final destination. VEGF, via downstream signaling molecules, such as extracellular signal-regulated kinase-1/2 (ERK1/2), stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), p38MAPK, and phosphatidylinositol 3-kinase/Akt (PI3K/Akt), is involved in cell survival, differentiation and migration. In response to external stimuli, activation of these mitogen-activated protein kinases (MAPKs) and PI3K/Akt cascade, in turn, activates intracellular signaling molecules and/or induces crucial alterations in several cytoskeleton-related proteins that are essential for cell migration. VEGF has been described to participate in the regulation of NSC migration. Evidence shows that the proper migration of neuronal progenitors along rostral migratory stream as well as olfactory bulb neurogenesis in adult mice is dependent on endogenous VEGF expression. Damage to the nervous system results in the increased VEGF expression, which induces the migration of neural stem cells into the area of infarction. More importantly, in vitro studies demonstrate that VEGF can guide the directed migration of NSCs, highlighting the critical role of this growth factor in neurogenesis during development and neural regeneration after injury.Cell migration, which requires coordinated reorganization of the actin cytoskeleton and regulation of cell-adhesion dynamics, is critical for a variety of biological processes in normal and pathological conditions including cellular development, tissue repair, and migration. VEGF could induce the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and then regulates vinculin and talin recruitment to focal adhesions leading to the speading of cells. Phosphorylated paxillin enhanced lamellipodial protrusions, whereas non-phosphorylated paxillin was essential for fibrillar adhesion formation and for fibronectin fibrillogenesis. Importantly, Paxillin-null fibroblasts display abnormal focal adhesion resulting in the impaired cell migration, indicating that paxillin palys an important role on the regulation of migration of cells.The recent findings that NSCs implanted ipsilaterally or contralaterally in the injured brain display a strong tendency to migrate toward pathological insults raise hope to develop new therapeutic strategies for CNS injury repair. However, it has been reported that a significant proportion of the transplanted NSCs do not demonstrate pathology-tracking capacities. This is confirmed by observations indicating that only a small subpopulation successfully reached the damaged brain areas, leading to a very low rate of cell replacement. Consistent with this note, there is evidence showing that VEGF stimulates migration of NSCs from subventricular zone-derived neurospheres and these migrating cells are comprised of a specific subpopulation of NSCs or progenitors that are immunoreactive for GFAP and nestin. Together, these data suggest that tropism for pathology in brain is likely to exhibit by a subpopulation of NSCs that are at a specific differentiation stage. We thus speculate that chemotactic responses are different among cells at different differentiation states and cells at a certain level of differentiation may be endowed with stronger migratory capacity or chemotactic responsiveness than cells at other differentiation states. To test this hypothesis, using the well-characterized murine NSC line C17.2, which was derived from the external germinal layer of the postnatal cerebellum, we analyzed the chemotactic responses of NSCs to concentration gradients of VEGF in relation to their differentiation states. Some findings are as followings: The plasticity of differentiation of NSCs A neural stem cell line C17.2 cells was chosen to prepare subpopulatioins of differentiating NSCs and to study the relationship between differentiation and migration of NSCs. Our immunocytochemical characterizations revealed that undifferentiated NSCs expressed nestin, but did not display antigenic markers for neuron- or glia-restricted precursor cells, including A2B5, GFAP,β-III tubulin and NSE. Upon differentiation, these NSCs gradually possessed the early cell-lineage specific markers for neurons or glial cells, however, there was still a loss of expression of NSE by 3 d. Further, we switched these differentiating cells to serum-containing medium and found that these cells not only morphologically changed to NSCs after maintained for 48 h but owned the proliferation capacities, suggesting that cells at 12 h, 1 d, and 3 d of differentiation we used in the present study are just at the early neural differentiation stage, or a transition state of the neurogenic differentiation while maintaining the potential to give rise to neurons and glia.The migratory capacity of NSCs towards VEGF depends on the differentiating stages of these cells To assess the directional migration of NSCs under different differentiation states in vitro, using a microchemotaxis Boyden chamber, we tested the transfilter migration of these cells toward VEGF at concentrations ranging from 5 ng/ml to 100 ng/ml. Results show the chemotactic response of undifferentiated NSCs to VEGF at 25 ng/ml and 50 ng/ml, but not at 5 ng/ml and 100 ng/ml. By contrast, VEGF at 5 ng/ml was observed to stimulate chemotaxis of differentiating NSCs and cells that had been differentiated for 1 d and 3 d, but not for 12 h, underwent chemotaxis when exposed to VEGF at 100 ng/ml. The peak chemotactic response occurred when VEGF at 25 ng/ml (1.68-fold), 5 ng/ml (2.16-fold), 25 ng/ml (3.18 fold) or 50 ng/ml (3.16 fold) was used for undifferentiated cells (cells of 0-h differentiation), cells of 12-h, 1-d and 3-d differentiation, respectively. These data indicate that the degree of the chemotactic responses and the migratory capacity of NSCs towards VEGF vary greatly, depending on the differentiating stages or states of these cells. NSCs at certain differentiation states possess stronger chemotactic response than cells at other differentiation states Data above suggest that the chemotactic responses to VEGF are different among cells at different differentiation states. We thus speculate that cells at a certain level of differentiation may be endowed with stronger migratory capacity or chemotactic responsiveness than cells at other differentiation states. To test this hypothesis, we then examined the response of NSCs under different differentiation states to the certain concentration of VEGF. The strongest chemotactic migration appeared in cells of 1-d differentiation, with 3.2 fold induction as compared to unstimulated cells, demonstrating that NSCs of 1-d differentiation possess stronger chemotactic response than cells at other differentiation states. Then we used the directed-viewing Dunn chamber to detail the migratory repinses of NSCs to VEGF. Quantitative analyses revealed that NSCs of 0-h, 12-h and 1-d differentiation showed significant increase in FMI, indicating the increase in the migration efficiency toward VEGF in these cells. Importantly, cells of 1-d differentiation showed a maximum directional persistence, which is consistent with the above observations showing that NSCs of 1-d differentiation possess the strongest chemotactic response in transfilter migration. Further, using primary NSCs isolated from the subventricular zone of newborn rat brains and expanded in FGF-2, we verified the VEGF-induced migration of these cells with respect with their differentiation. Consistent with the abovementioned observations, results showed a close relationship between the migratory behavior that were described by the migration speed and the migration efficiency, and the differentiation states. While a decrease in migration speed was found in the process of differentiation, significantly decreased at 40-h compared with cells of 4- and 16-h differentiation, the migration efficiency displayed a biphasic time-dependent curve with a maximum directional persistence in cells of 16-h differentiation, confirming that the migratory response of NSCs to VEGF correlates closely with their differentiation states.The responses of MAPKs, PI3K/Akt, the expression and distribution of paxillin were different among cells at different differentiation states after exposed to VEGF Following stimulation, a significant increase of Akt phosphorylation was produced and remained elevated until 12 h ar undifferentiated and differentiating cells. Likewise, a sustained phosphorylation of ERK1/2 was also present in undifferentiated and differentiating NSCs. However, treatment with VEGF resulted in an increased phosphorylation with a maximum activation occurring at 5 min. By contrast, no obvious change in p38MAPK phosphorylation was detected in undifferentiated and differentiating NSCs with an exception that a transient increase in p38MAPK phosphorylation with a maximum activation occurring at 15 min and a return to basal level by 30 min was found in cells of 3-d differentiation. While the basal SAPK/JNK phosphorylation was very low, an increase of SAPK/JNK phosphorylation was observed, which was significant from 5 min to 30 min in undifferentiated cells, from 5 min to 1 h in cells of 12-h differentiation. SAPK/JNK phosphorylation subsequently declined and reached the low basal level with the exception in cells of 1-d and 3-d differentiation in which a sustained elevated phosphorylation lasted until 12 h. Meanwhile, the expression and the distribution area of paxillin were increased after exposed to VEGF 5 min in undifferentiated cells. By contrast, this phenomenon was present at 1 min in cells of 12-h and 1-d differentiation, while there was no change in cells of 3-d differentiation after exposed to VEGF. These data confirmed that the responses of MAPKs, PI3K/Akt, the expression and distribution of paxillin were different among cells at different differentiation states after exposed to VEGFThe PI3K/Akt and MAPKs pathway are involved in VEGF-induced migration of differentiating NSCs NSCs were pretreated with the inhibitor of MAPKs or PI3K/Akt prior to their exposure to the gradient of VEGF added to the lower well of a microchemotaxis Boyden chamber at the concentration of 25 ng/ml. Results showed that interference with PI3K/Akt signaling by LY294002 prevented NSCs of 12-h differentiation from migrating in response to VEGF gradient, while no effects were observed on undifferentiated cells and cells of 1-d and 3-d differentiation. Inhibition of ERK1/2 signaling significantly attenuated VEGF-stimulated migration of both the undifferentiated and differentiating NSCs and conversely, NSCs showed normal chemotactic response after treatment with inhibitors of SAPK/JNK or p38MAPK. These data indicate that while ERK1/2 signaling participates in the regulation of directed migration or chemotaxis of both undifferentiated and differentiating NSCs, PI3K/Akt signaling only plays a role in cells at certain differentiation state(s).To further assess the mechanisms that regulate the VEGF-induced migration of differentiating NSCs, Dunn chamber was used to directly observe the migratory behavior of differentiating NSCs in the presence of inhibitors. In accordance with the results above, compared with untreated NSCs that chemotactically migrate towards VEGF, inactivation of ERK1/2 signaling by PD98059 led to a deficit of chemotaxis of both undifferentiated and differentiating NSCs, with a decreased migration speed and FMI. A reduction of FMI was also observed in cells of 12-h and 1-d differentiation upon exposure to PI3K/Akt inhibitor, which didn’t change FMI of undifferentiated cells and cells of 3-d differentiation, or the migration speed of all cells tested. Intriguingly, although we found that inhibiting SAPK/JNK or p38MAPK signaling had no effects on the transfilter migration of NSCs towards VEGF, time-lapse video analysis revealed that the inhibitor of p38MAPK suppressed the speed of cells of 0-h, 12-h and 1-d differentiation and the migration efficiency, i.e. FMI, of cells of 12-h, 1-d and 3-d differentiation and that inhibition of SAPK/JNK by SP600125 led to a decreased migration speed in cells of 0-h and 1-d differentiation, with no changes in FMI of all NSCs and in migration speed of cells that had differentiated for 12 h and 3 d, implying that SAPK/JNK and p38MAPK signaling may contribute to the control of the migration speed and the migration efficiency, but not the establishment of directionality of NSCs in response to VEGF.Neural regeneration is a complex process that involves the proliferation of NSCs, the sensing of the surrounding factors, the migration of these cells to the lesion sites, and their differentiation into the localized cell types. It is unlikely that the in vivo migration of NSCs, a prerequisite event for the regeneration of the injured tissues, is regulated by only one factor. In fact, many cytokines, including VEGF, stem cell factor, stromal-derived factor, and monocyte chemoattractant protein-1, are found to be involved in the regulation of NSC migration. Results presented here raise the intriguing possibility that NSCs at certain differentiation state might possess stronger capacity of chemotaxis than undifferentiated NSCs and NSCs at other states, highlighting a pivotal role of the differentiation states in the chemotactic responses of NSCs. The transplanted NSCs at certain differentiation state might promote the structural and functional repair of the damaged brain more efficiently than do undifferentiated NSCs and NSCs at other levels of differentiation, which is of great importance in the context of repair after brain injury. Similar phenomena were observed with NSCs stimulated by stromal-derived factor-1alpha, hepatocyte growth factor, stem cell factor, or platelet-derived growth factor, implying that this might represent a general rule in chemotactic responses of NSCs. More intensive studies are required to identify these responsive NSCs, and to delineate the cellular and molecular mechanisms that govern the directed migration of these cells, thereby allowing for optimization of the therapeutic potential of NSCs to be employed for neural regeneration after injury.