Department of Pediatric Neurosurgery, Children's Hospital of Fudan University, Shanghai, People's Republic of China.
Cell Mol Neurobiol. 2012 Apr;32(3):373-80. doi: 10.1007/s10571-011-9765-3. Epub 2011 Dec 15.
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.
本研究旨在从鼠海马神经干细胞中获得高纯度的星形胶质细胞培养物，并将其与从富含混合神经胶质的培养物中分离出的星形胶质细胞进行比较，后者通常被小胶质细胞污染。我们制备了新生鼠海马神经干细胞的原代培养物，这些细胞具有分化为星形胶质细胞、神经元和少突胶质细胞的潜能。我们通过有限稀释法产生单克隆神经干细胞集落。我们通过在聚 L：-赖氨酸上接种集落并在含有 10%胎牛血清和 100ng/ml 睫状神经营养因子的诱导培养基中培养来诱导星形胶质细胞分化。然后，我们通过差异粘附和在恒定温度下摇动对细胞进行进一步纯化，然后进行第二轮有限稀释。胶质纤维酸性蛋白的免疫细胞化学染色表明，我们的方法得到了 99.4±0.5%纯度的星形胶质细胞，而传统的富集混合神经胶质培养物得到了 94.2±2%纯度的星形胶质细胞。诱导细胞在功能特性方面类似于原代星形胶质细胞培养物，例如细胞增殖率、无致瘤性和 p53 缺失，以及表皮生长因子受体、bystin 和一氧化氮合酶的表达。因此，我们的神经干细胞培养和纯化的新方法可常规用于从鼠海马中获得高纯度星形胶质细胞的原代培养。
