Establishment of embryonic neural stem cell clone in rats following ordinal treatment of basic fibroblast growth factor and epidermal growth factor

ABSTRACT

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.

To investigate the effective methods to culture neural stem cells in vitro using culture medium containing different growth factors.

Single sample observation.

Laboratory of Cell Biology, College of Life Science, Sichuan University.MATERIALS Ten SD rats which were pregnant for 14 days, DMEM/F12 11, 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.

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 (11 )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

① Primary clone of neural stem cells.② Clone culture result of unicell. ③ Induction and differentiation 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.

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.