The Study Of Neural Stem Cells From Olfactory Mucosa Tissue Of Human

ObjectiveTo establish the method for cultruing neural stem cells(NSCs) from olfactory mucosa of human, and induce neural stem cells to differentiate into neurons, so that fundamental research data can be provided for cultruing neural stem cells(NSCs) from olfactory mucosa of human, and new research object can be provided for the study of nervous system disease.Methods1.Wash the biopsies in DMEM/HAM F12 containing 1%streptomycin/penicillin several times to wipe off blood.2.Slice the biopsies into 3–4 pieces with a thickness ranging from 200 to 500 μm in a sterile culture dish. Then wash these biopsies in DMEM/HAM F12 containing 1% streptomycin/penicillin again.3.Incubate these biopsies in fetal bovine serum for 15 min. And then insert each biopsies into its own 2 cm diameter culture dish, which has been smeared with fetal bovine serum and cover the tissue with sterile 1.3cm diameter glass cover slips. In the first 24 hours, add a little DMEM/F12 containing 10 % FBS and 1 % streptomycin/penicillin to soak the biopsies.Incubating biopsies in fetal calf serum was aimed to make the biopsies adhere to the culture dish easier.4.Add 300 ul of culture medium(DMEM/HAM F12 supplemented with 10% fetal calf serum and 1% streptomycin/penicillin) to each culture dish after the first 24 hours. Renew the culture medium every 2 days.5.Seven days later, stem cells will begin to invade the culture dish and after two weeks they should be confluent. When confluency is reached,passage and transfer the cells to the larger culture dishes.6.Prepare flasks by incubating them for 2h at 37 °C with poly-L-lysine(1–5 μg/cm2).7.Passage the olfactory cells using trypsin and plate the cells at a density of 15,000 cells/cm2 in the treated flasks.8.Feed the cells with the sphere inducing culture medium(DMEM/F12 supplemented with insulin, transferrin(1%), EGF(50ng/ml),FGF2(25–50ng/ml), and 1% streptomycin/penicillin).9.collect floating cell spheres from the medium change.10. To differentiate olfactory stem cells into neuron-like cells,cultivate them for 5 days in Neurobasal medium containing B-27,penicillin/streptomycin and glutamate.11.Then make medium changes every 3 days. Neuron-like cells should appear after two weeks.ResultsOlfactory stem cells are dividing rapidly and confluency can be reached within two weeks. Two key features of stem cells—sphere formation and nestin expression—were evaluated. When grown on poly-l-lysine with a serum-free culture medium supplemented with insulin,transferrin(1%), EGF(25ng/ml), FGF-2(50 ng/ml), and 1%streptomycin/penicillin, olfactory stem cells gave rise to spheres. We also observed that the olfactory stem cells expressed nestin. When grown in a Neurobasal culture medium supplemented with B27 and glutamate, most of the nasal olfactory stem cells differentiate into neuron-like cells expressing MAP2.Conclusion1. We successfully obtained neural stem cells which were Nestin immunoreactive by cultruing olfactory mucosa of human.2. These cells can be induced to neurospheres. When grown in a Neurobasal culture medium supplemented with B27 and glutamate, the neurospheres can differentiate into neuron-like cells.3.In general, we successfully obtained neurol stem cells from olfactory mucosa of human, and got neurons from these neural stem cells.These results shed some lights on the study of the study of nervous system disease.