BACKGROUND: Adult central nervous system lacks the ability to regenerate, so it is of great significance to find a new source of neural stem cells.
OBJECTIVE: To investigate the differences between basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in inducing bone marrow mesenchymal stem cells (MSCs) to differentiate into neurons in vitro.
METHODS: MSCs were isolated from normal human bone marrow using density gradient centrifugation and cell attachment method. MSCs were plated in 96-well culture plates at a density of 0.25×108/L and cultured with 200 μL DMEM/F12 for 0, 1, 2, 3, 4, 5, 6, and 7 days, with 20 μL MTT (5 mg/mL) in 5 wells at each time point. The supernatant was removed and 100 μL dimethyl sulphoxide was added to each well for 4 additional hours of incubation. In addition, some cells were exposed to bFGF and EGF. Growth curve was determined with MTT method. Telomerase activity were examined by TRAP(PCR)-ELISA. Additionally, the functional differences of the two cytokines were checked by RT-PCR.
RESULTS AND CONCLUSION: RT-PCR revealed that nestin, glial fibrillary acidic protein (GFAP) and neurofilament subunit M (NF-M) mRNA were expressed in un-induced MSCs of passage 4. Nestin expression reduced at 7 days. The expression of micro-tubule-associated protein-2 (MAP2) mRNA was not detected until the induction, and increased thereafter. The expression of MAP2 mRNA was greater in bFGF+EGF and bFGF alone groups compared with EGF alone group, and the expression of GFAP in EGF alone group was greater than other groups. Results showed that MSCs can be cultivated, proliferated and differentiated into neural stem cells in vitro. The differentiated neural stem cells have the activity of proliferation, but not have the ability of infinite proliferation as tumor cells.