使用胚体冰冻切片分析的多能干细胞
Summary
多能干细胞悬浮液中生长分化成胚状体（EBS）。在这里，我们展示了如何获得高品质的EB冰冻切片，用于研究胚胎发育的细胞和分子方面，同时保留其作为集合体的组织。
Abstract
胚胎干细胞（ES细胞）产生的多能干细胞的囊胚阶段的早期哺乳动物胚胎的内细胞团。在ES细胞分化的一个关键阶段，是形成胚状体（EBS）集合2， 3。 EB的形成是基于自发聚集在非贴壁板培养的ES细胞。三维的EB概括许多方面的早期哺乳动物胚胎发育和分化成三个胚层：外胚层，中胚层和内胚层4。
免疫荧光和原位杂交技术被广泛用于目标在一个组织的第5，6，7细胞中的蛋白质和mRNA的检测技术。在这里，我们提出一个简单的技术来产生高质量的冰冻切片胚体。这种方法依赖于EB嵌入在华侨城的空间方向的cryosection技术。由此产生的部分可以受到各种各样的分析程序，以表征含有的某些蛋白质，RNA或DNA的细胞种群。在这个意义上说，EB的冰冻切片的准备（10微米），是必不可少的工具（如Hematoxilin和曙红，DAPI）的组织学染色分析，免疫荧光法（如OCT4，巢）或原位杂交。这种技术还可以帮助了解胚胎发育方面与关于维护三EBS的三维球形结构。
Protocol
1。固定和冷冻保存丝裂霉素C灭活，并辅以20％的血清替代淘汰赛（KSR）和8ng /毫升的成纤维细胞生长因子（FGF – 2）的DMEM/F12维持到小鼠胚胎成纤维细胞（MEF）的多能干细胞培养。为了诱导EB形成H9的细胞被转移到非贴壁的菜肴和7天，保持在15％KSR 8为辅的DMEM/F12培养。 （！）注意：这种技术可以用于任何胚胎和诱导多能干细胞衍生分化。 用移液器从培…
Discussion
这里介绍的方法提供了一个易于遵循协议来获取煤灰固定薄低温恒温器的部分有用的胚体免疫荧光法和原位杂交检测。造成冰冻切片允许人类胚胎干细胞分化的细胞和分子方面的研究，同时保持它们的结构和组织作为聚合。
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是保护行使宪法权利Fundação支持Pesquisa做州里约热内卢（FAPERJ），Conselho国立发展Científico发送Tecnológico圣保罗大学和研究所的全国Ciência发送Tecnologia（INCTC）。布鲁纳保尔森和艾琳M.费尔南德斯的EB图像，我们非常感谢。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| D (+) Sucrose | Reagent | Vetec | 228 | |
| Paraformaldehyde | Reagent | Rieden-de Haën | 16005 | |
| PBS solution | Reagent | LGC Biotecnology | 13-30259.05 | |
| Poly-L-lysine hydrobromide | Reagent | Sigma | P2636 | 200mg/mL in water |
| Tissue-Tek O.C.T. Compound | Reagent | Sakura | P2636 | |
| Sucrose Solution | Reagent | 10% Sucrose Solution in PBS w/v | ||
| Sucrose Solution | Reagent | 20% Sucrose Solution in PBS w/v | ||
| Sucrose Solution | Reagent | 30% Sucrose Solution in PBS w/v | ||
| Conic Tube | Tool | TPP | 91015 | 15mL conic tube |
| Plate shaker | Tool | Biomixer | ||
| Cryostat | Tool | Leica | CM 1850 | |
| Mold for OCT platform | Tool | Plastic mold plataform |
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Tags
Pluripotent Stem CellsCryosectionsEmbryoid BodiesES CellsDifferentiationAggregatesEctodermMesodermEndodermImmunofluorescenceIn Situ HybridizationTarget ProteinsMRNACryosection TechniqueAnalytical ProceduresHistology Staining AnalysisHematoxilin And EosinDAPIOct4NestinEmbryogenesisTri-dimensional Spherical Structure
Cite This Article
Gomes, I. C., Acquarone, M., Maciel, R. d. M., Erlich, R. B., Rehen, S. K. Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies. J. Vis. Exp. (46), e2344, doi:10.3791/2344 (2010).