BACKGROUND: The effective method to obtain neural stem cells through in vitro culture that deserves to pay more attention in experimental studies of stem cells.OBJECTIVE: To investigate the effective methods to culture neural stem cells in vitro using culture medium containing different growth factors.DESIGN: Single sample observation.SETTING: Laboratory of Cell Biology, College of Life Science, Sichuan University.MATERIALS: Ten SD rats which were pregnant for 14 days, DMEM/F12 1:1, basic fibroblasts growth factors, epidermal growth factor; nestin antibody IgG , β- microtubule protein Ⅲ antibody IgG , glial fibrillary acidic protein antibody IgG, biotin labeled second antibody and third antibody and fluorescinisothiocyate (FITC)-labeled second antibody were used in this experiment.METHODS: This experiment was carried out at the Laboratory of Cell Biology, College of Life Science, Sichuan University from 1999 to 2001. ① Brain tissue was taken out from the procerebrum of fetal rat, then primary neural stem cell clone was obtained through enzymatic digestion, mechanical treatment, centrifugation and culture. ② Neural stem cells were inoculated and cultured at 2×108 L-1 and divided into 4 groups with 6 bottles of cells in each group. DMEM/F12 (1:1 )culture medium containing 0.1 volume fraction of fetal bovine serum was added , serving as DMEM/F12 group; culture medium containing 20 μg/L basic fibroblast growth factor was added , serving as basic fibroblast growth factor group; culture medium containing 30 μg/L epidermal growth factor was added , serving as epidermal growth factor group; Culture medium containing basic fibroblast growth factors was added to culture for 2 hours, then culture medium containing epidermal growth factors was used for further culture, serving as basic fibroblast growth factor+ epidermal growth factor group. Culture flask was change after 24-hour culture; another 14 days later, primary clone number was counted under the microscope, and expressions of specially labeled albumen nidogen of stem cells were detected with immunohistochemistry.MAIN OUTCOME MEASURES: ① Primary clone of neural stem cells.② Clone culture result of unicell. ③ Induction and differentiation results.RESULTS: ① The cells isolated from the brain of fetal rats possess the ability to consecutively passage and form clone , and immunofluorescent staining showed cell nidogen expression positive in the cell sphere. ②Clone rate of neural stem cells was the highest (0.630%)using ordinal culture of basic fibroblast growth factor and epidermal growth factor. ③ The cultured neural stem cell clone can be induced and differentiated neurons and glinl cells.CONCLUSION: ① The results proved that the cultured and isolated cells are neural stem cells. ②Ordinal treatment of basic fibroblast growth factor and epidermal growth factor is an effective method to obtain neural stem cells.

This is a study on the cultivation condition in vitro and differentiation of neural stem cells from human embryonic brain in order to find a way to get purified multipotential neural stem cells. The single cells was derived from the three-month embryonic brain digested with trypsin, some cells was frozen, the other cells were expanded with EGF and bFGF, the single-cell-clone was obtained by the way of limited dilution, and the serum was used to induce the cells differentiation. The cells were detected with the method of immunohistochemistry. The results showed that a lot of neurospheres could be seen in the presence of mitogens (both EGF and bFGF) and serum could induce neural stem cells to differentiate into neurons, astrocytes, and oligodendrocytes. These indicate that the survival and proliferation of neural stem cells rely on the cooperation of EGF and bFGF. The neural stem cells can also be harvested from the frozen cells.
Astrocytes ; cytology ; Brain ; cytology ; embryology ; Cell Differentiation ; Cells, Cultured ; Culture Media ; chemistry ; Epidermal Growth Factor ; chemistry ; Fibroblast Growth Factor 2 ; chemistry ; Humans ; Immunohistochemistry ; Neural Stem Cells ; cytology ; Neurons ; cytology ; Oligodendroglia ; Pluripotent Stem Cells ; cytology

In this study, we collected human umbilical cord blood and separated the autologous serum and clear cells. Then the human mesenchymal stem cells were cultured with the use of autologous serum and fetal serum, respectively. By identifing surface markers with flow cytometry and testing the osteogenic, adipogenic and cardiomyocytes differentiation capacity of the cells, we affirmed that the cultured cells are mesenchymal stem cells. Among 21 cases of umbilical cord blood, 11 cases using fetal bovine serum and 15 cases using autologous successfully cultured mesenchymal stem cells from umbilical cord blood. The UCB-MSCs did not express CD34 and CD45, but they expressed CD29, CD44 and CD105, and they were successfully induced into osteogenic cells, adipogenic cells and cardiomyocytic-like cells. All of these suggest that using autologous serum to culture UCB-MSCs is better than using fetal bovine serum, and the autologous serum cultured UCB-MSCs can also be induced into osteogenic cells, adipogenic cells cardiomycyote-like cells.
Cell Differentiation ; physiology ; Cell Division ; Cells, Cultured ; Culture Media ; Fetal Blood ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Serum