| BACKGROUND AND PURPOSES:Neural stem cells have the capacity for continued proliferation and self-renewal and can differentiate into neurons, astrocytes, oligodendrocyte. Furthermore, they can provide a new source of a donor for the reconstruction of the central nervous system function and nerve regeneration. They also help to resolve the ethical issues and shortage of donors for the neural transplantation and therefore bring new hopes for the treatment of many central nervous diseases such as Parkinson's disease.Anterior subventriculor zone is widely recognized as a neural stem cell-rich region. Stimulated by dorsal- ventral signal, the neural stem cells move towards the olfactory bulb (OB) along the Rostral migratory stream (RMS), and finally differentiate in the olfactory bulb into the intermediate neurons with OB phenotype. These intermediate neurons include dopaminergic neurons andγ-aminobutyric acid neurons.In addition to self-renewal capacity and multiple differentiation potential like regular stem cells, SVZa neural stem cells also have the following characteristics: (a) have the potential of development into neurons at the moment when they are produced; (2) can migrate directionally in a long distance. (3) always maintain proliferation capacity and the potential of neuronal differentiation during the relocation process without further differentiation. Therefore, SVZa neural stem cells have become the best model for the study of proliferation, migration and differentiation of neural stem cells.Neural stem cell differentiation mainly depends on two factors: First, inherent genetic information of stem cells; Second, the microenvironment of stem cells. Stem cells of the same sources can differentiate into different types and species of cells in different microenvironment. Thus, besides their inherent gene regulation, current studies of stem cells focus on the regulation of external signals. As a member of the transforming growth factor-β(TGF-β) superfamily, BMP2 is a very important cytokine in the local microenvironment of stem cells. How BMP2 promotes the differentiation of SVZa neural stem cells into dopaminergic neurons in vitro still needs further experimental research.Furthermore, previous studies showed that induction of differentiation of neural stem cells into dopaminergic neurons by a single factor is very limited. Hence, it is necessary to explore the effects of multiple factor combination, thereby increasing the percentage of differentiation of SVZa stem cells into dopaminergic neurons. As an in vitro cell culture medium for astrocytes, ACM (Astrocyte-conditioned medium) contains abundant soluble bioactive substances secreted by astrocytes such as nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). The medium is very close to the in vivo microenvironment of neural stem cells and has an incomparable function in promoting differentiation when compared to a single neurotrophic factor. Therefore, the goals of this study are: First, to isolate SVZa neural stem cells; Second, to induce differentiation of SVZa neural stem cells with different concentrations of BMP2 and measure the corresponding percentage of differentiation into dopaminergic neurons. Finally, to explore the roles of BMP2 and ACM in the differentiation of neural stem cell into dopaminergic neurons. Hopefully these studies will provide some exciting experimental data to the research on the directional differentiation of SZVa neural stem cell.METHODS :1. Cell culture technology was used to isolate and culture SVZa neural stem cells. The cultured clones were subjected to immunocytochemical identification of Nestin. Immunocytochemical identification of NF, GFAP and CNP was also performed for the differentiated clones to determine if they have characteristics of stem cells and multiple differentiation potential.2. Cultured SVZa neural stem cells were incubated with different concentrations of BMP2 ( 0, 0.1, 1, 10, 20, 100 ng / ml) to be induced to differentiate. Immunohistochemical staining and flow cytometry were used to measure the corresponding percentage of differentiation of SVZa neural stem cells into TH+(tyrosine hydroxylation enzyme) neuron.3. Cultured SVZa neural stem cells were treated with vehicle, BMP2, ACM or BMP2 + ACM to be induced to differentiate. Immunohistochemistry and flow cytometry were performed to analyze the percentage of differentiation of neural stem cells into TH+ cells. RESULTS :1. In serum-free culture conditions, we obtained a great number of suspended Nestin-positive cell clones. Immunocytochemistry results reveal that those clones are also positive for NF, GFAP and CNP after differentiation.2. After SVZa neural stem cells were treated with 0, 0.1, 1, 10, 20 and100 (ng / ml) concentrations of BMP2, the cells incubated with 0.1～100 (ng/ml) of BMP2 show a significantly higher percentage of TH + cells than untreated cells (P<0.05). It peaks at 10ng/ml.3. Cultured SVZa neural stem cells were treated with vehicle, BMP2, ACM or BMP2 + ACM to be induced to differentiate. Immunohistochemistry and flow cytometric analysis suggest that the experimental groups produce much higher percentage of TH + cells than control group. The group treated with BMP2+ACM has the highest percentage of TH+ cells. The difference is statistically significant (P<0.05).CONCLUSIONS:1. We successfully isolated neural stems cells from the SVZa brain tissue of rats at day 16 of pregnancy. The isolated SVZa neural stem cells have constant proliferation and self-renewal capacity and can differentiate into neurons, astrocytes and oligodendrocytes.2. BMP2 can promote differentiation of SVZa neural stem cells into dopaminergic neurons. However, the induction of differentiation is not entirely in a dose-dependent manner and there is an optimal concentration. 10ng/ml of BMP2 produces the most significant effect.3. ACM can promote differentiation of SVZa neural stem cells into dopaminergic neurons. BMP2 has a synergistic effect with ACM on the induction of differentiation. |
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