Abstract:

BACKGROUND: The application of embryo or brain-derived neural stem cells has been greatly hampered by ethical and moral constraints or technical difficulties. 
OBJECTIVE: To culture SD rat bone marrow-derived neural stem cells in vitro using serum-free neural induction medium.
METHODS: Bone marrow mesenchymal cells (BMSCs) were collected from rat femurs and tibias, which were isolated and cultured by the whole bone marrow culture and adherence method. The cell cycle and immunophenotype were detected by flow cytometry, and the osteogenic and adipogenic potential were identified by oil red O staining and alizarin red staining. The 4-6 generations of BMSCs were incubated to differentiate into bone marrow-derived neural stem cells under serum-free medium containing epidermal growth factor, basic fibroblast growth factor and B27, followed by DMEM/F12 supplemented with fetal calf serum, the differentiated cells were identified by immunofluorescence and flow cytometry.
RESULTS AND CONCLUSION: (91.5±3.1)% of the P5 BMSCs were in G1 phase, which highly expressed CD90 and CD29, but not expressed CD45 and CD34. Reddish yellow lipid droplet could be seen after osteogenic induction, and black mineralized nodules could be found after adipogenic induction. Bone marrow-derived neural stem cells were positive to nidogen immunofluorescence staining, and the positive rate was (97.2±1.1)%. The cells expressed neuron specific enolase, β-Tubulin, glial fibrillary acidic protein as well as microtubule-associated protein-2 antigen. It demonstrated that BMSCs can differentiate into neural stem cells under the induction of serum-free neural induction medium combined with certain growth factors. And the bone marrow-derived neural stem cells have the ability of differentiation into neurons, astrocytes or oligodendrocytes.