Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube
Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube
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DOI：
10.3791/4134
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发表时间：
2012-06-01
影响因子：
1.2
通讯作者：
Labosky, Patricia A.
中科院分区：
文献类型：
--
作者：
Pfaltzgraff, Elise R.;Mundell, Nathan A.;Labosky, Patricia A.
胚胎神经嵴（NC）是一种多能祖细胞群，起源于神经管的背侧，经历上皮细胞至间充质转化（EMT）并迁移到整个胚胎，产生不同的细胞类型（1-3）。NC还具有影响靶器官的分化和成熟的独特能力（4-6）。当在体外外植时，NC祖细胞进行自我更新、迁移并分化为多种组织类型，包括神经元、神经胶质、平滑肌细胞、软骨和骨。NC多能性首先从鸟类神经管的外植体中描述（7-9）。NC细胞的体外分离有利于NC动力学的研究，包括增殖，迁移和多能性。在鸟类和大鼠系统中的进一步研究表明，当移植回胚胎时，经灭活的NC细胞保留其NC潜力（10-13）。由于这些固有的细胞特性被保存在体外培养的NC祖细胞中，因此神经管外植体法为体外研究NC提供了一个有吸引力的选择。NC衍生的祖细胞可以从胚胎和成体的迁移后位置培养，以研究迁移后NC祖细胞的动力学（11，14 -20），然而，当NC祖细胞从神经管迁移时，分离NC祖细胞提供了NC细胞潜能和迁移特性的最佳保存（13，21，22）。一些方案采用荧光激活细胞分选（FACS）来分离富集特定祖细胞的NC群体（11，13，14，17）。然而，当从早期胚胎开始时，用FACS难以获得足够用于分析的细胞数量，使得从单个胚胎分离早期NC群体变得复杂。在此，我们描述了一种不依赖于FACS的方法，该方法基于Wnt 1-Cre激活的谱系报告基因产生约96%纯的NC群体（23）。与以前的方法相比，该方案的优点是：1）细胞不在饲养层上生长，2）不需要FACS来获得相对纯的NC群体，3）分离预迁移的NC细胞，以及4）结果容易定量。此外，该方案可用于分离NC从任何突变的小鼠模型，促进NC的特性与不同的遗传操作的研究。这种方法的局限性在于NC从胚胎的环境中去除，已知这影响NC 2的存活、迁移和分化，24-28。
The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types (1-3). NC also has the unique ability to influence the differentiation and maturation of target organs(4-6). When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone.NC multipotency was first described from explants of the avian neural tube(7-9). In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo(10-13). Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro.To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors(11,14-20), however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties(13,21,22). Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors(11,13,14,17). However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter(23).The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.