A Modified In vitro Method to Obtain Pure Astrocyte Cultures Induced from Mouse Hippocampal Neural Stem Cells Using Clonal Expansion

摘要

The aim of the present study was to produce astrocyte cultures of high purity from mouse hippocampal neural stem cells and to compare their in vitro properties with those isolated from enriched mixed glial cultures prepared from mouse hippocampus, which are commonly contaminated by microglia. We produced primary cultures of newborn mouse hippocampal neural stem cells, which have the potential to differentiate into astrocytes, neurons, and oligodendrocytes. We produced monoclonal neural stem cell colonies by limiting dilution. We induced astrocyte differentiation by plating the colonies on poly-l-lysine and culturing them in induction medium consisting of minimum essential medium/F12 supplemented with 10% fetal bovine serum and 100 ng/ml ciliary neurotrophic factor. We then further purified the cells by differential adherence and shaking at a constant temperature, followed by a second round of limiting dilution. Immunocytochemistry for glial fibrillary acidic protein showed that our method yielded 99.4 ± 0.5% pure astrocytes, whereas traditionally enriched mixed glial cultures yielded 94.2 ± 2% pure astrocytes. Induced cells resembled primary astrocyte cultures in functional properties such as cell proliferation rates and lack of tumorigenicity and p53, and expression of epidermal growth factor receptor, bystin, and nitric oxygen synthase. Our novel method of culture and purification of neural stem cells can therefore be used routinely for the primary culture of highly purified astrocytes from mouse hippocampus.