Neural Differentiation Of Mesenchymal Stem Cells Influences Their Chemotactic Responses To Hepatocyte Growth Factor

With the infiltrating nature of tumors, malignant gliomas are the most prevalenttype of primary brain tumors. Although extensive surgical excision with adjuvant radio-and chemo-therapy has been in a popular use, which does not fully meet our expectationTransplantation of progenitor cells is a promising approach for the treatment of gliomas.Bone marrow mesenchymal stem cells (BMSCs) are candidates for such a cell-basedtherapy, since they display an extensive tropism for gliomas and different chemotaticfactors (such as SDF, SCF, VEGF, HGF) in vitro and in vivo, and have the capacity todifferentiate into osteoblasts, chondrocytes and adipocytes. However, few datas areavailable on the factors involved in regulating BMSCs migration. The aim of our studywas to investigate the migration behavior of BMSCs under different differentiationstates towards HGF.BMSCs were isolated from bone marrow of rats by Percoll gradient centrifugationand expanded. The cells were characterized by morphology, antigen expression anddifferentiation potential. The results showed that BMSCs displayed fibroblast-like shapeand proliferated quickly. Immunofluorescence analysis showed that BMSCs werepositive for CD29, CD90, and CD106, and negative for CD34, CD45. Moreover,BMSCs were induced to differentiate into adipocytes and osteoblasts, demonstratingthat these cells are multipotential BMSCs.BMSCs were induced to neuron-like cells by basic fibroblast growth factor (bFGF)combination with butyl hydroxy anisol (BHA). The undifferentiated BMSCs werefibroblast-like. After preinductin in10ng/ml bFGF for24h, the edge of the cellsbecame rough. Cells after5-h induction in medium containing2%DMSO and200μM BHA displayed nucleus refractile cell bodies and long branching processes, with thecytoplasm retracted towards the nucleus. After maintaining in H-DMEM containing N2for48h, the processes continued to elaborate, displaying primary and secondarybranches, growth cone-like terminal structures that frequently made contact with othercells. Our immunocytochemical characterizations revealed that nestin-positive cellswere highest in undifferentiated stage. When cultures were allowed to differentiate ininduction medium, the induced cells began to express β-III tubulin. Upon differentiation,the number of cells expressing nestin decreased, whereas the number of cells expressingβ-III tubulin increased over time. In states of18-h and48-h maintenance, very few cellsstill expressed nestin, and the induced cells were weakly stained with β-III tubulin.However, there was still a loss of expression of MAP2by maintenance for48h.In this study, we performed Boyden chamber assays to test the migration of thesecells toward HGF at concentrations ranging from5ng/ml to100ng/ml.we proved HGFinduced BMSCs migration in a dose-dependent manner, with the maximum migrationobserved in50ng/ml added to the bottom compartment of the Boyden chamber.Meanwhile, we also investigated whether the observed migratory effect waschemotactic or chemokinetic. We find when identical amounts of HGF or L-DMEMwere added to both the top and the bottom compartments, cell migration of BMSCsunder different differentiation states remained unchanged. We further investigatedchemotaxis of BMSCs under different differentiation states (24-h preinduction,5-hinduction,18-h and48-h maintenance) toward HGF. Results showed HGF at50ng/mlwas observed to stimulate chemotactic migration of differentiating BMSCs, and theaddition of25ng/ml HGF to the lower chamber triggered a significant increase intransfilter migration of5-h induction,18-h and48-h maintenance. Furthermore, we alsoobserved the migratory effect of neural differentiating BMSCs toward HGF waschemotactic but not chemokinetic. In addition, we also investigated the migrationnumber of BMSCs under different differentiation states towards HGF, find that cells of24-h preinduction present more intensively chemotactic response than cells at otherdifferentiation states. We characterized the responsive cells that had migrated to the bottom side of the Boyden chamber by immunofluorescence staining and found that, atall differentiation stages, nestin-positive cells constituted the majority of the migratorycells. HGF induced a significant increase in the nestin-positive cells while no change ofthe transfilter migration of β-III tubulin-positive cells was observed. Consistent with ourobservations that cells of24-h preinduction showed the strongest chemotactic responseto50ng/ml HGF, the number of HGF-induced migrating cells expressing nestin in24-hpreinduction state was significantly higher than that of other differentiation statesFurthermore, we utilized a Dunn chamber in conjunction with time-lapse videoanalysis to directly observe and follow the HGF-induced migration of differentiatingBMSCs. The data recorded by Leica AF6000were analyzed by NIH Image J to getmigration speed and migration efficiency. Additionally, compared with cells of5-hinduction,18-h and48-h maintenance, cells at the state of undifferentiation and24-hpreinduction showed a significant increase in migration speed, however, no markeddifference was detected among them. Moreover, cells of24-h preinduction showed amaximum directional persistence, which is in accordance with our previousobservations showing that BMSCs of24-h preinduction trigger the strongestchemotactic response in transfilter migration.MAPKs are a family of protein kinases that contain ERK1/2, p38MAPKs, andSAPK/JNK, Akt is a known downstream mediator of the PI3K-dependent signalingcascade. Both PI3K/Akt and MAPKs signal transduction pathways have involved inmany kinds of bioeffects including mitosis, apoptosis, proliferation, differentiation andmigration by chemokines or cytokines in various cell types. It has been reported thatHGF activates downstream effectors, such as ERK1/2, PI3K and p38MAPK in variouscell types. To further investigate the involvement of PI3K/Akt and MAPKs signalingtransduction pathways in migration, we used Western blot to examine the effect of HGFon ERK1/2, Akt, p38MAPK and SAPK/JNK phosphorylation in BMSCs under differentdifferentiation states. Firstly, undifferentiated BMSCs were treated with severalconcentrations of HGF (0-100ng/ml) for30minutes. Stimulation with HGF led to aconcentration-dependent phosphorylation of ERK1/2, Akt and p38MAPK, but not SAPK/JNK. To futher study the effects of HGF on these two pathways, we use50ng/mlHGF to treat BMSCs under different differentiation ststes for various times. The resultsindicated that BMSCs under different differentiation states all expressed a basal level ofphosphorylation of Akt, ERK1/2, p38MAPK and SAPK/JNK, however, the increasedphosphorylation of Akt for HGF-treatment was only observed in undifferentiation,24-hpreinduction and5-h induction states. In cells at undifferentiated and24-h preinduction,the HGF-induced ERK1/2phosphorylation was significantly elevated from5min,peaking at15min and virtually returned to basal levels within30min. The sustainedtime of ERK1/2phosphorylation was prolonged to about60min in cells of5-hinduction,18-h maintenance and48-h maintenance states. Treatment with HGF resultedin a transient increase in p38MAPK phosphorylation at5min and peaking at15min incells of undifferentiation,24-h preinduction and5-h induction. However, no significantchanges were detected in18-h and48-h maintained MSCs. In all the times of HGFtreatment, SAPK/JNK phosphorylation states remained unchanged in undifferentiatedand24-preinduced cells, but were observed to be transiently increased at5min in cellsof5-h induction, and increasingly elevated from5min to15min in cells of18-hmaintenance and15min to60min in cells of48-h maintenance. Further, we detectedthe basal level expression of ERK1/2, Akt and p38MAPK and SAPK/JNK in BMSCsunder different differentiation states. We found that the expression levels of ERK1/2,Akt, p38MAPK and SAPK/JNK are similar in the five states cells respectively, however,their phosphorylation (p-ERK1/2, p-Akt, p-p38MAPK, p-SAPK/JNK) levels varied indifferent differentiation stages. Compared with cells of undifferentiation,24-hpreinduction and48-h maintenance, cells at the states of5-h induction and18-hmaintenance showed even elevated phosphorylation expression of ERK1/2. Moreover,Akt phosphorylation expression in undifferentiated BMSCs was greater than otherdifferentiated states cells. However, phosphorylated p38MAPK and SAPK/JNK weregradually increased.Cytoskeletal rearrangement is closely associated with the cell migration. Migrationrequires a cell polarization toward a chemoattractant, and the rate of cell migration has been attributed to the capability of a cell to reorganize its cytoskeleton. We thereforeanalyzed the organization of the cytoskeleton using immunofluorescence detection ofF-actin at various times after HGF-stimulation. Immunocytochemical analysis of thedistribution of F-actin in HGF-treated or untreated BMSCs revealed that, at the states ofundifferentiation and24-h preinduction, a large number of cells did transiently formactin-rich lamellopodia at the edge of them, and showed some retracting uropodsandsfrom0.5min of stimulation with50ng/ml HGF, whereas in cells at the states of5-hinduction,18-h and48-h maintenance, HGF significantly induced cytoskeletalreorganization from1.5min or so. In this study, we detected the changes of F-actinpolymerization and found that upon neural differentiation, the reorganization ofcytoskeleton became relatively insensitive to HGF stimulation.In conclusion, we performed the induction of BMSCs into neuron-like cells, andthrough Boyden chamber and Dunn chamber, analyzed the contribution of HGF tomigration of BMSCs under differentiation states. We demonstrated that HGF at variousconcentrations induced different chemotactai responses of BMSCs under differentdifferentiation states. Additionally, we used western blot and inhibitor experiments toinvestigate the relative functional role of PI3K/Akt and MAPK signaling transductionpathways in HGF-stimulated migration of BMSCs under differentiation states.HGF-induced actin polymerization is also closely related to the differentiation levels ofMSCs. The present study would contribute to better understanding of the directedmigration of BMSCs and thus, help design strategies for the clinical applications ofBMSCs.