The Effect Of SCF On The Migration Of Bone Marrow Mesenchymal Stem Cells In Neurogenic Differentiation

Objective Malignant gliomas are primary brain tumors which are hurmful to the health of people. Bone marrow mesenchymal stem cells (BMSCs) are candidates for a cell-based therapy, since they display an extensive tropism for gliomas,besides it is easy to gain. However few or nobody to investigate the tropism of BMSCs in different differentiation states, for this, the migration behavior of BMSCs in neurogenic differentiation towards stem cell factor (SCF) is studied in this paper.Methods (1) BMSCs were isolated by Percoll gradient centrifugation from rat, cultivated and amplified, and phenotypically identified by immunofluoresce staining. (2) BMSCs were induced to differentiate into neural-like cells by the following steps: cells were treated with 10 ng/mL basic fibroblastic growth factor (bFGF) for 24 h, followed by 200μM butyl hydroxy anisole (BHA) plus 2% dimethylsulfoxide (DMSO) for 5 h. Finally, cells were maintained with DMEM (H) plus N2 for 48 h. We observed the morphology of BMSCs during neurogenic differentiation with inverted microscope, and the expression of neural markers, nestin,β-III-tubulin and NSE were analyzed by immunocytochemistry. (3) We investigated, by using the Dunn chamber, the directional migration of BMSCs in neurogenic differentiation towards the concentration gradients of SCF. The migration of differentiated cells was followed by Leica AF6000, and analyzed by NIH Image J. Then statistics was done to get migration speed, migration efficiency and migration trajectory. The effect of LY294002, the inhibitor of PI3K, on the BMSCs migration induced by SCF was also studied by Dunn chamber.Results (1) We isolated and cultured rat bone marrow mesenchymal stem cells by using Percoll lymphocyte separation and discontinuous density gradient method. Immunofluorescence analysis showed that BMSCs were positive for CD29, CD90, CD106, and negative for CD34, Besides, they can be induced to differentiate into osteocytes and adipocyte. (2) BHA, bFGF and DMSO were used to induce the neural differentiation of BMSCs, after pre-induced 24 h, BMSCs became spindle. Treatment of BMSCs with BHA for 5 h led to a dramatic change in morphology, such as cell body contraction and neural-like axis production. Then maintained 48 h, cells took on more bifurcation and secondary bifurcation, while no changes were found in the control group. The expression of neural markers, nestin,β-III-tubulin and NSE were found in the cells differentiated by immunocytochemistry. (3) Dunn chamber analysis revealed that both the migration speed and efficiency of cells induced by SCF were signifficantly higher than control, demonstrating the chemotactic effect of SCF on BMSCs. BMSCs at different differentiation states showed different tropisms for SCF. Besides, the adding of LY294002 depressed the migration speed and efficiency.Conclusion BMSCs can be induced to differentiate into neuron-like cell; Tropism of BMSCs for SCF was closely related to their differentiation states. PI3K participates in this process.