Department of Micro- and Nanotechnology, Technical University of Denmark, Lyngby, Denmark.
PLoS One. 2013 Aug 27;8(8):e73669. doi: 10.1371/journal.pone.0073669. eCollection 2013.
Neurospheres are used as in vitro assay to measure the properties of neural stem cells. To investigate the molecular and phenotypic heterogeneity of neurospheres, molecular beacons (MBs) targeted against the stem cell markers OCT4 and SOX2 were designed, and synthesized with a 2'-O-methyl RNA backbone. OCT4 and SOX2 MBs were transfected into human embryonic mesencephalon derived cells, which spontaneously form neurospheres when grown on poly-L-ornitine/fibronectin matrix and medium complemented with bFGF. OCT4 and SOX2 gene expression were tracked in individual cell using the MBs. Quantitative image analysis every day for seven days showed that the OCT4 and SOX2 mRNA-expressing cells clustered in the centre of the neurospheres cultured in differentiation medium. By contrast, cells at the periphery of the differentiating spheres developed neurite outgrowths and expressed the tyrosine hydroxylase protein, indicating terminal differentiation. Neurospheres cultured in growth medium contained OCT4 and SOX2-positive cells distributed throughout the entire sphere, and no differentiating neurones. Gene expression of SOX2 and OCT4 mRNA detected by MBs correlated well with gene and protein expression measured by qRT-PCR and immunostaining, respectively. These experimental data support the theoretical model that stem cells cluster in the centre of neurospheres, and demonstrate the use of MBs for the spatial localization of specific gene-expressing cells within heterogeneous cell populations.
神经球被用作体外测定以测量神经干细胞的特性。为了研究神经球的分子和表型异质性，设计了针对干细胞标志物 OCT4 和 SOX2 的分子信标 (MBs)，并以 2'-O-甲基 RNA 骨架合成。OCT4 和 SOX2 MBs 转染到人胚胎中脑衍生细胞中，当在聚-L-鸟氨酸/纤维连接蛋白基质和补充有 bFGF 的培养基上生长时，这些细胞会自发形成神经球。使用 MBs 跟踪单个细胞中的 OCT4 和 SOX2 基因表达。在分化培养基中培养的神经球的每日定量图像分析显示，OCT4 和 SOX2 mRNA 表达细胞聚集在神经球的中心。相比之下，分化球体边缘的细胞发育出神经突并表达酪氨酸羟化酶蛋白，表明发生了终末分化。在生长培养基中培养的神经球包含分布在整个球体中的 OCT4 和 SOX2 阳性细胞，并且没有分化的神经元。MBs 检测到的 SOX2 和 OCT4 mRNA 的基因表达与 qRT-PCR 和免疫染色分别测量的基因和蛋白质表达密切相关。这些实验数据支持了干细胞聚集在神经球中心的理论模型，并证明了 MBs 可用于在异质细胞群体中对特定基因表达细胞进行空间定位。
