Balic Anamaria
Research Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Methods Mol Biol. 2019;1922:29-37. doi: 10.1007/978-1-4939-9012-2_4.
Continuous growth of the rodent incisor is enabled by epithelial and mesenchymal stem cells (ESCs and MSCs) which unceasingly replenish enamel and dentin, respectively, that wear by persistent animal gnawing. Lineage tracing studies have provided evidence that ESCs contribute to all epithelial lineages of the tooth in vivo. Meanwhile, in the mouse incisor, MSCs continuously contribute to odontoblast lineage and tooth growth. However, in vitro manipulation of ESCs has shown little progress, mainly due to lack of appropriate protocol to successfully isolate, culture, expand, and differentiate ESCs in vitro without using the co-culture system. In this chapter we describe the isolation of the Sox2-GFP+ cell population that is highly enriched in ESCs. Isolated cells can be used for various types of analyses, including in vitro culture, single cell-related analyses, etc. Furthermore, we describe ways to obtain populations enriched in the incisor MSCs using FACS sorting of antibody-labeled cells. Easily accessible FACS sorting enables easy and relatively fast isolation of the cells labeled by the fluorescent protein.
啮齿动物的门齿持续生长是由上皮干细胞和间充质干细胞（ESCs和MSCs）实现的，它们分别不断补充因动物持续啃咬而磨损的牙釉质和牙本质。谱系追踪研究已经提供证据表明，上皮干细胞在体内对牙齿的所有上皮谱系都有贡献。同时，在小鼠门齿中，间充质干细胞持续对成牙本质细胞谱系和牙齿生长有贡献。然而，上皮干细胞的体外操作进展甚微，主要是因为缺乏在不使用共培养系统的情况下成功分离、培养、扩增和分化上皮干细胞的合适方案。在本章中，我们描述了高度富集上皮干细胞的Sox2-GFP+细胞群体的分离方法。分离出的细胞可用于各种类型的分析，包括体外培养、单细胞相关分析等。此外，我们描述了使用抗体标记细胞的荧光激活细胞分选（FACS）来获得富集在门齿间充质干细胞群体的方法。易于操作的FACS分选能够轻松且相对快速地分离出由荧光蛋白标记的细胞。
