一种酶和无血清神经干细胞培养模型EMT研究适合药物发现
1Dept. of Biomedicine, Pharmacenter,University of Basel, 2Molecular Signalling and Gene Therapy,Narayana Nethralaya Foundation, Narayana Health City, 3Brain Ischemia and Regeneration, Department of Biomedicine,University Hospital Basel, 4Department of Neurosurgery,Klinikum Idar-Oberstein, 5Department of Neurosurgery and Institute for Stem Cell Biology and Regenerative Medicine,Stanford University, 6Department of Neurology, Laboratory of Molecular Neuro Oncology,University Hospital of Zurich
Summary
Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.
Abstract
上皮至间质转变（EMT）描述的上皮转分化成间充质的过程。 EMT是也通常发生在胶质母细胞瘤，最常见的恶性脑肿瘤胚胎发育过程中的基本处理。 EMT也已在脑外多种癌包括乳腺癌，肺癌，结肠癌，胃癌观察。 EMT是通过促进迁移，侵袭和转移形成集中挂恶性肿瘤。 EMT诱导的机制尚未完全清楚。在这里，我们描述了皮层神经干细胞（NSCs）和随后的EMT诱导的标准化隔离的体外系统。这个系统提供了以使用单细胞或外植体培养的灵活性。在这个系统中，大鼠或小鼠胚胎前脑的神经干细胞是在限定的培养基中培养，不含血清和酶。的神经干细胞表达Olig2的和SOX10两个转录因子在oligodendroc观察YTE前体细胞（OPCs的）。使用这个系统，FGF-，BMP-和之间的相互作用的TGFβ信号传导涉及ZEB1，ZEB2，观察TWIST2哪里的TGFβ-活化显著增强细胞迁移，这表明有协同BMP-/TGFβ-相互作用。结果指向FGF-，BMP-和TGFβ-信令网络，以参与EMT诱导和维持。这个模型系统相关的体外研究EMT。它是具有成本效益的和显示出高再现性。它也允许对不同的化合物相对于它们的迁移响应（定量距离测量），和化合物抑制或增强EMT（定性测定）的高通量筛选的比较。因此，该模型是非常适合于测试药物库影响EMT物质。
Introduction
在胚胎发育的几个阶段，上皮细胞失去强粘附到彼此（ 例如，紧密连接），并获得在这个过程被称为上皮至间质转变（EMT）1迁徙表型。 EMT需要形成额外的细胞类型，如间充质神经嵴细胞，一个人口从神经上皮2偏析。 EMT是不在胚胎阶段只有必要的，但还需要在成年生命的后期阶段保持在成年生物体的生理过程，如伤口愈合3和中枢神经系统（CNS）的再生在脱髓鞘病灶4。
上皮性肿瘤是众所周知的激活EMT作为迁移，侵袭和转移引发步骤，最终导致癌症的发展1,3。 EMT确实集中连接到强大的迁移1,3。 C的细胞的步骤onditioning，发起，接受和维持EMT尚不完全清楚，需要进一步调查。
在此，提出了一种基于神经干细胞的标准化体外 EMT模型系统，以确定生长因子和媒体（无血清和无酶的使用）。这个模型系统是在EMT工作的科学家的相关性。蜗牛，瑞伯和Twist蛋白家族已被证明是既在发育和疾病1为EMT的关键。蜗牛，瑞伯和扭曲的家庭也参与所提出的系统。该系统是基于通常不经历EMT提供一个特别的优点为初始事件的EMT诱导时研究的前脑的特定区域。
该模型系统可能被用于研究在CNS外上皮EMT，由于关键的EMT诱导剂，如蜗牛，瑞伯和扭曲的蛋白质，也都EMT期间在CNS外组织系统中发现的。该型号System使神经干细胞的分离标准化来自发展中国家皮质学习一般干细胞的功能和EMT尤其如此。使用该系统，我们分离的神经干细胞，诱导的EMT和研究FGF2和BMP4的作用下随后的迁移。我们观察到，FGF-和BMP-信令相互作用与TGFβ-信令以促进细胞迁移，从而验证该模型的系统。
Protocol
所有动物的程序遵循指南“实验动物的护理和使用”（NIH出版， 第 8版，2011）和巴塞尔的动物福利委员会（用于动物的护理和使用瑞士准则）获得批准。通过这些准则的动物协议被认为是“最严重的动物品位”的。 1.膨胀培养基的制备注：在无菌条件下的标准组织培养工作。 取两个15毫升管，加入5 ml L-谷氨酰胺的DMEM / F12（1：1）从500毫升培养基瓶到每个两个15毫升管中…
Representative Results
此EMT模型系统是基于对NSCs都为单细胞或作为从显影神经管的特定区域的外植体的标准化的隔离，中央皮质（ 图1和2）。对于量化考核，在植500微米格培养皿中（ 图3）的中心接种的权利。从中央皮质植体首先暴露于FGF2两天，随后在生长因子（ 表1）的不同组合的其他两天。我们开始与它比显影神经管的其它区域更大的皮?…
Discussion
在这项研究中描述了EMT的分析利用神经干细胞的标准化系统（总结于补充图3）。标准化保证再现性（ 表1和2）。的神经干细胞被从显影皮质衍生的，通常不经历EMT的组织。这是有利的为在EMT早期步骤的分析。在EMT初始步骤不能充分研究中已积累的遗传改变，并且可以已经通过EMT的特征的肿瘤细胞。此外，原发肿瘤的样品是不理想的理解EMT因为大多数恶性肿瘤是异质含有?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究是由一个赠款MHS和AG（SNF IZLIZ3_157230）巴塞尔科学基金会的大学和瑞士国家科学基金会的支持。我们感谢：塔尼亚里纳尔迪博士Burkat慷慨地提供基础设施;该Bettler小组讨论和评论的所有成员。我们感谢多恩格哈德（徕卡公司，瑞士）的专业和称职的安装全高清MC170摄像机（徕卡公司，瑞士）。
Materials
| BMP4, rhBMP4 | RnD Systems | 314-BP-01M | |
| Bovine pancreas insulin | Sigma | I1882 | |
| Boyden chamber, CytoSelect cell invasion assay | Cell Biolabs | CBA-110 | 24 well plate system |
| Cell culture dish with grid | Ibidi 500 mm dish, 35 mm | 80156 | |
| CellMask Orange | Life Technologies | C10045 | Plasma membrane dye, use at 1:1000 . |
| DAPI | LifeTechnologies | D1306 | Stock at 5mg/ml. Use at 1:10000. Cancerogenic. Appropriate protection (gloves, coat, goggles) required. |
| DMEM/F12 1:1 medium bottle | Gibco Invitrogen | 21331-020 | |
| FGF2, rhFGF2 | RnD Systems | 233-FB-01M | |
| Fibronectine, bovine | Sigma | F4759 | |
| Glutamax supplement | Gibco Invitrogen | 35050-061 | |
| Graphics software with pixel measurement feature | Fiji | fiji.sc/Fiji | version 2.0.0-rc-30/1.49s |
| HBSS media | Sigma | H9394 | |
| Human apo-Transferrin | Sigma | T1147 | Possible lung irritant. Avoid inhalation. Use appropriate protection. |
| L-glutamine | Gibco Invitrogen | 25030-024 | |
| Nestin, Mouse anti Nestin antibody | Genetex | GTX26142 | Use at 1:100, 4% PFA fixation, Triton X100 at 0.1% |
| Olig2, Rabbit anti Olig2 antibody | Provided by Hirohide Takebayash | Personal stock | Use at 1:2000, 4% PFA fixation, Triton X100 at 0.1% |
| Penicillin/Streptomycin/Fungizone | Gibco Invitrogen | 15240-062 | |
| Podoplanin, Mouse anti Podoplanin antibody | Acris | DM3614P | Use at 1:250, 4% PFA fixation, avoid Triton X100 |
| Poly-L-ornithine | Sigma | P3655 | |
| Putrescine | Sigma | P5780 | Skin and eye irritant. Appropriate protection required. |
| Sodium selenite | Sigma | S5261 | |
| Sox10, Rabbit anti Sox10 antibody | Millipore Chemicon | AB5774 | Use at 1:200, 4% PFA fixation, Triton X100 at 0.1% |
| TGFb1, rhTGFb1 | RnD Systems | 240-B-010 | |
| Uncoated Petri dishes | Falcon Corning | 351029 |
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Citer Cet Article
Sailer, M. H. M., Sarvepalli, D., Brégère, C., Fisch, U., Guentchev, M., Weller, M., Guzman, R., Bettler, B., Ghosh, A., Hutter, G. An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery. J. Vis. Exp. (114), e54018, doi:10.3791/54018 (2016).