Fortunel Nicolas O, Vaigot Pierre, Cadio Emmanuelle, Martin Michèle T
Laboratoire de Génomique et Radiobiologie de la Kératinopoïèse, CEA, DSV, Evry, France.
Methods Mol Biol. 2010;585:13-23. doi: 10.1007/978-1-60761-380-0_2.
The basal layer of human interfollicular epidermis is thought to contain a minor compartment of quiescent or slowly cycling epithelial stem cells. These primitive keratinocytes give rise to the progenitors, which are the proliferating keratinocytes and which can be defined as early to late progenitors, according to their differentiation status. Because of the intrinsic heterogeneity of the basal layer, the development of new methods suitable for functional analysis of basal keratinocytes directly isolated from skin samples is greatly needed. We describe here a new method that allows a rapid and multiparallel deposition of single keratinocytes into 96-well plates, using flow cytometry. The first step of the process allows the clonal analysis of the growth potential of freshly isolated epithelial cells in primary cultures. In a second step, various techniques of functional characterization can be performed on the progeny of the cloned cell, including the generation of reconstructed epidermis, colony assays, and secondary cloning. In a third step, a long-term characterization of the progeny of the cloned keratinocytes can be performed, either by successive subclonings or mass expansion cultures.
人们认为，人毛囊间表皮的基底层含有一小部分静止或缓慢循环的上皮干细胞。这些原始角质形成细胞产生祖细胞，即增殖的角质形成细胞，根据其分化状态可将其定义为早期至晚期祖细胞。由于基底层存在内在异质性，因此迫切需要开发适用于直接从皮肤样本中分离的基底角质形成细胞功能分析的新方法。我们在此描述一种新方法，该方法可利用流式细胞术将单个角质形成细胞快速、多平行地接种到96孔板中。该过程的第一步允许对原代培养中新鲜分离的上皮细胞的生长潜力进行克隆分析。第二步，可以对克隆细胞的后代进行各种功能表征技术，包括重建表皮的生成、集落测定和二次克隆。第三步，可以通过连续亚克隆或大规模扩增培养对克隆角质形成细胞的后代进行长期表征。
