Tropism Of Differentiating Mesenchymal Stem Cells For HGF

Due to the infiltrating nature of tumors, the treatment of malignant gliomas invariantly fails despite extensive surgical excision and adjuvant radio- and chemo- therapy. Therefore, new therapeutic strategies are needed to selectively target tumor cells, including those that have escaped from the main tumor mass. Mesenchymal stem cells (MSCs) are adult stem cells with the capacity to differentiate into osteoblasts, chondrocytes and adipocytes. In addition, the migratory capacity of MSCs towards gliomas and several chemotatic factors has made these cells attractive therapeutic tool in gene therapy for gliomas. However, little is known about the migratory capacity of MSCs with respect to their differentiation states. The aim of our study was to characterize the migratory behavior of bone marrow mesenchymal stem cells (BMSCs) during neurogenic differentiation in response to hepatocyte growth factor (HGF).BMSCs were isolated from bone marrow of rats by Percoll gradient centrifugation and expanded. The cells were characterized by morphology, antigen expression and differentiation potential. The results showed that BMSCs displayed fibroblast-like shape and proliferated quickly. Immunofluorescence analysis showed that BMSCs were positive for CD29, CD90, and CD106, and negative for CD34, CD45. Moreover, BMSCs were induced to differentiate into adipocytes and osteoblasts, demonstrating that 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 were fibroblast-like. After preinductin in 10 ng/ml bFGF for 24 h, the edge of the cells became rough. Cells after 5-h induction in medium containing 2% DMSO and 200μM BHA displayed nucleus refractile cell bodies and long branching processes, with the cytoplasm retracted towards the nucleus. After maintaining in H-DMEM containing N2 for 48 h, the processes continued to elaborate, displaying primary and secondary branches, growth cone-like terminal structures that frequently made contact with other cells. Immunofluorescence staining showed that the expression of neural stem cell marker nestin and early state neuronal markerβ-III-tubulin increased and peaked at 5-h induction, and then decreased significantly, while the ratio of cells expressing NES remained low before maintaining period and increased remarkably after maintaining for 48 h. The cells were constantly GFAP negative during differentiation.In this study, we utilized a Dunn chamber in conjunction with time-lapse video analysis to directly observe and follow the HGF-induced migration of differentiating BMSCs. The data recorded by Leica AF6000 were analyzed by NIH Image J to get migration speed and migration efficiency. The chemotaxis analysis, in which the outer well was filled with 50 ng/ml HGF and the inner well with L-DMEM only, showed that HGF did not change the migration speed of BMSCs, while the migration efficiency of BMSCs increased gradually with the increasing concentration of HGF. In contrast, neither the migration speed nor the migration efficiency was changed in the chemokinesis experiment in which both inner and outer wells were filled with 50 ng/ml HGF. The data indicate that HGF can induce the directed migration of BMSCs by chemotaxis. Next, we analyzed the chemotactic migration of BMSCs in neurogenic differentiation in response to the concentration gradients of HGF. The migration assay showed that HGF at 50 ng/ml did not change the migration speed of the cells at any state in neurongenic differentiation. However, HGF elevated the migration efficiency of undifferentiated BMSCs, and BMSCs pre-induced for 24 h, induced for 5 h and maintained for 48 h, but not those maintained for 18 h. The results indicate that the directed migration of BMSCs towards HGF is closely related to their differentiation states. To explore the role of PI3K signaling pathway in the tropism of BMSCs for HGF, the cells were pretreated for 1 h with LY294002 (30μM), a specific inhibitor of PI3K, and then used for chemotaxis assay. The data showed that the migration efficiency decreased significantly compared with untreated cells, indicating that PI3K participates in the directed migration of BMSCs to HGF.To determine the role of Rac1 in HGF-induced migration, we altered the Rac1 expression in BMSCs and then analyzed the migratory behavior of these cells. The results showed that both the migration speed and migration efficiency were increased by up-regulation of Rac1, while decreased by down-regulation of Rac1, suggesting that Rac1 regulates the HGF-induced migration of BMSCs.In conclusion, the isolated BMSCs from bone marrow by Percoll gradient centrifugation are multipotential and capable of differentiating into neuron-like cells by bFGF combination with BHA. HGF could induce the directional migration of BMSCs, which is mediated by the PI3K signaling pathway. In addition, the migration of cells during neurogenic differentiation towards HGF is closely related to their differentiation states. Rac1 regulates the migration speed and participates in the HGF-induced migration of BMSCs. The present study would contribute to better understanding of the directed migration of BMSCs and thus, help design strategies for the clinical applications of BMSCs.