从成年小鼠切牙牙源性上皮干细胞的分离和培养
1Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology,University of California, San Francisco, 2Department of Bioengineering and Therapeutic Sciences,University of California, San Francisco, 3Department of Pathology and Research Center,Zhongshan Hospital of Dalian University, 4Université Paris Descartes, Sorbonne Paris Cite, UMR S872, 5Centre de Recherche des Cordeliers,Université Pierre et Marie Curie, UMR S872, 6INSERM U872, 7Division of Endodontics, Department of Preventive and Restorative Dental Sciences,University of California, San Francisco, 8Department of Pediatrics and Institute for Human Genetics,University of California, San Francisco
Summary
不断增长的鼠标切牙提供了一个模型，以研究牙源性上皮干细胞（DESCs）牙齿组织的重建。一个强大的系统，从切牙持续，可靠地获得这些细胞，并扩大它们在体外这里报道。
Abstract
了解背后牙齿再生和更新的细胞和分子机制已成为极大的兴趣1-4个主题，鼠标切牙提供了一个模型，这些过程。这一显着的器官在整个动物的生命持续增长，并产生所有从容纳在阴唇（朝向唇）和舌（朝向舌）颈环（CL）的区域的成体干细胞的活性池所需的细胞类型。只有从阴唇CL中的牙齿干细胞生成搪瓷，牙齿的外壳，在唇面釉细胞产生。这种不对称的釉质形成允许磨损在切牙尖，和祖细胞和干细胞在近端门齿确保牙齿组织正在不断地补充。隔离和生长这些祖细胞或干细胞在体外的能力允许其扩张和打开大门，大量的实验不能达到体内 ，如H潜在的干细胞调控因子的IGH吞吐量测试。在这里，我们描述并演示从小鼠切牙的唇CL可靠和一致的方法来培养细胞。
Introduction
之一的脊椎动物的独特特征是牙齿的演化。牙齿已成为许多研究领域一个重要的模型系统，作为分子途径和相关与这个器官形态特化进行了研究从几个方面，包括发展和进化生物学家5。最近，再生医学领域已经开始使用牙作为一种模式来获得有价值的见解。尤其，牙科上皮干细胞的发现是一个重要的进步6-13。
所有啮齿类动物具有不断增长的门牙，其增长是得益于干细胞，使这些牙齿可访问的模型系统来研究成体干细胞的调控。标记实验在20世纪70年代10,11，接着遗传谱系追踪实验8,9,12,14，已经证明，DESCs驻留在门牙的近侧区域。干细胞的后代在唇侧移动了公认的小生，被称为唇颈环（CL）的上皮车厢，并有助于细胞称为运输扩增（TA）细胞（ 图1）的人口。具体来说，DESCs驻留在外部釉上皮（OEE）和星状网（SR）8,9,14，和内釉上皮细胞（IEE）产生了对TA的细胞通过细胞周期的数量有限的进步，然后移动远侧沿着门齿的长度（ 图1）。在小鼠中切牙成釉细胞分化继续向远侧移动沿切牙以约350微米15,16一天一个惊人的速度。因为他们移动，细胞分化为成熟的成釉细胞和中间层（SI）的细胞。沉积釉质基质的整个厚度后，许多成釉细胞发生凋亡，而其余的细胞收缩的尺寸和调节搪瓷成熟<燮> 17。在唇侧发光，如SR，其他类型的细胞谱系是不太清楚，并就干细胞在间充质18和舌CL数据才刚刚开始出现。
使用鼠标切牙模型，一些团体一直在努力阐明遗传途径和参与自然干细胞为基础的器官重建细胞生物学过程。但是，阴唇CL包含相对少量的细胞，估计为约5000每只小鼠切牙，这使得与原代细胞具有挑战性的工作。因此，已作出努力，以文化，以打开新的大门，以实验方法是无法达到在体内扩大这些细胞在体外 6,16,19,20。最近的研究表明，这些细胞可以既自我更新和分化成釉原蛋白表达细胞的培养时，13。在这里，我们描述和展示为次的方法E可靠和一致的从鼠标唇发光细胞的培养。
Protocol
1，解剖下半侧下颌骨从成年小鼠在此之前执行此协议，获得必要的机构批准，并一定要遵守所有的动物护理指南。使用标准IACUC批准程序安乐死的动物。对这项工作的CO 2窒息用于颈椎脱位。 可选：使用剃刀刀片的身体的其余部分分离头。 从下唇除去皮肤的领口。 使用手术刀，使两个下门牙，它们松散地连接之间的切口。当施加压力时，下颌骨将被分?…
Representative Results
鼠标半侧下颌骨包含一个不断增长的门牙三根臼齿（ 图1A）。所有的牙齿由牙本质和牙釉质，牙冠的两个矿化成分（ 图1A和1A'）的。门齿装有两个干细胞小生境称为CL唇和舌CL和搪瓷是在唇侧（ 图1A'）完全形成。 DESCs负责门齿釉质形成与被容纳在阴唇CL，特别是OEE和SR（ 图1A'）8,9。唇CL也包含独立外部评价，TA细胞，?…
Discussion
上皮细胞首次成功培养了40年前21-24，以及最近的上皮干细胞25-27成功分离了我们先进的上皮生物学知识。我们报告一个协议，用于分离成年小鼠切牙的DESCs，干细胞的相对充分研究人口，有产生了重要的见解牙科生物学和牙釉质形成的可能性。该协议最初是基于对上皮干细胞的分离从毛囊28以前的报告。而许多协议使用饲养层和血清维持上皮干细胞，我们的方法是一项联接…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢王秀萍的初步帮助与DESC文化。提交人由来自美国国立卫生研究院（K99-DE022059到AHJ，K12-GM081266到MGC，K08-DE022377-02 OH和R01-DE021420到ODK）奖学金和助学金资助了一部分。
Materials
| Forceps, size 5 | Fine Science Tools | 11251-30 | FST by Dumont, Dumoxel, |
| Forceps Straight, fine, 3.5 | Roboz | RS5070 | |
| Razorblades | Electron Microscopy Services | #71960 | |
| Scalpel Handle No.3 | VWR | ||
| Feather Blade No. 15 | Electron Microscopy Services | #72044-15 | Surgical Stainless Steel |
| Collagenase Type-1 filtered | Worthington Biochemical Corporation | #4214 | |
| Insulin syringe | BD | #329424 | |
| Accumax | EMD Millipore | #SCR006 | |
| DMEM/F12 | Gibco | 11320-033 | |
| EGF | R&D | 2028-EG-200 | |
| FGF2 | R&D | 233-FB-025 | |
| B27 Supplement | Gibco | 10889-038 |
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Cite This Article
Chavez, M. G., Hu, J., Seidel, K., Li, C., Jheon, A., Naveau, A., Horst, O., Klein, O. D. Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor. J. Vis. Exp. (87), e51266, doi:10.3791/51266 (2014).