人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析
Summary
本文详细介绍了在培养人类胎儿脑神经干细胞的方法, 以及如何将它们区分成各种神经元亚型和星形胶质细胞, 重点是利用神经干细胞研究 Zika 病毒感染。
Abstract
人胎脑神经干细胞是一种独特的非基因修饰的模型系统, 研究各种刺激对人类发育神经生物学的影响。而不是使用动物模型或基因修饰的诱导多能细胞, 人类神经干细胞提供了一个有效的体外系统, 以检查治疗效果, 筛选药物, 或检查个体差异。在这里, 我们提供了详细的协议, 用于扩展人类胎儿脑神经干细胞在无血清培养基中的培养, 通过不同的启动程序将它们分化成各种神经元亚型和星形胶质细胞, 并进行冷冻和恢复这些细胞。此外, 我们描述了一个使用人胎脑神经干细胞研究 Zika 病毒感染的过程。
Introduction
Zika 病毒 (ZIKV) 是一种黄传播的性行为, 或由蚊子媒介的伊蚊蚊和白纹伊蚊蚊子。ZIKV 最近被认为是一个严重的公共卫生威胁, 由于其易于传播和附属神经系统症状1。其中一个最有关的神经效应是发育的小头在胎儿出生的孕妇感染的母亲2,3。小头是一种神经发育障碍, 在胎儿发育和出生时, 其头部小于典型的大小, 周长低于平均4的2标准偏差。较小的头围通常伴有各种并存, 如发育迟缓、癫痫、视力和听力丧失以及喂养困难。
最近的研究使用动物模型或诱导多能干细胞来研究 ZIKV 感染对神经的影响5,6,7,8。虽然这些研究有助于我们对 ZIKV 的认识, 但使用不同物种或基因修饰的细胞可能会耗费时间和/或增加可能混淆 ZIKV 对开发神经细胞的影响的其他变量5,6,7,8. 然而, hNSC 文化的困难, 特别是本协议中描述的非粘附干细胞文化, 是文化对用于进行区域性9的方法非常敏感。培养基成分的任何变化, 甚至培养容器的物理处理, 都足以引起细胞9的反应。为了解决这些问题, 我们开发了一个体外人胎儿脑源性神经干细胞 (hNSCs) 培养机械询问 ZIKV 对胎儿神经干细胞的影响。使用我们的方法, hNSCs 保持超过80通道没有明显的表型变化10。此外, 染色体的改变, 如体是无或最小的11。这种 hNSC 文化成长为一种非附着的干细胞文化。球的一个优点是, 球体在文化中创造了一个独特的环境生态位, 比起二维区域性9更能反映在体内的利基。该协议的另一个优点是可以从 hNSC 文化中获得多种细胞类型, 从而使调查人员能够观察给定变量对 hNSC 生存和分化的影响。本议定书适用于寻求回答有关中枢神经系统发育或功能障碍的机械问题的个人。下面的协议描述了如何扩大 hNSC 的文化感染与 ZIKV, 并随后区分 hNSCs, 以观察感染的影响, 分化过程。它还包括存储 hNSCs 用于长期使用的方法, 并将 hNSCs 分化成各种类型的神经元, 以便进一步调查 ZIKV 引起的大脑畸形的缺陷11。我们相信, 这项议定书也有兴趣, 调查人员试图了解任何环境刺激, 如感染或毒素对神经干细胞的生存和分化的影响。
Protocol
Representative Results
Discussion
培养和操纵 hNSCs 的能力提供了一个关键的工具, 可用于各种目的从建模人类疾病到高通量药物筛选10,11,12,14, 15、16、17。许多问题仍有待解决, 如人胎脑神经干细胞或其子代如何易受 ZIKV 感染, 不同的 ZIKV 菌株是否会以同样?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了来自邓恩基金会和德克萨斯大学医学分会 (p.w) 人类感染和免疫研究所的资助。
Materials
| DMEM | Gibco | 11965-092 | |
| F12 | Gibco | 11765-054 | |
| Glucose (10%) | Sigma | G8644 | |
| HEPES (1M) | Corning/cellgro | 25-060-c1 | |
| Pen Strep (100x) | Gibco | 15140-122 | |
| Insulin | Sigma | I4011 | |
| L-Glutamine | Gibco | 25030-081 | |
| Transferrin | Sigma | T2036 | |
| Progesterone | Sigma | P8783 | |
| Putrescine | Sigma | P5780 | |
| Sodium selenite | Sigma | S5261 | |
| Heparin Sigma | Sigma | H3149 | |
| basic fibroblast growth factor | R&D system | 233-FB | |
| epidermal growth factor | R&D system | 236-EG | |
| leukemia inhibitory factor | R&D system | 7734-LF | |
| Laminin | Invitrogen | 23017-015 | |
| 2.5% trypsin | Gibco | 15090-046 | |
| Trypsin inhibitor | Sigma | T6522 | |
| Poly-D-Lysine hydrobromide(PDL) | Sigma | P6407-5MG | |
| B-27 supplement | Gibco/Invitrogen | 17504-044 | |
| Fetal bovine serum | Gibco | 16000-044 | |
| Dimethyl sulfoxide | Sigma | D2650-100ml | |
| Dulbecco's phosphate-buffered saline | Corning/cellgro | 21-031-CV | |
| Bovine serum albumin | Sigma | A4378-25G | |
| Normal goat serum | Jackson ImmunoResearch Lab | 005-000-121 | |
| Triton X-100 | FisherBiotech | BP151-500 | |
| Nestin antibody | BD Transduction Laboratories | 611659 | 1:200 dilution |
| Class III beta-tubulin antibody (TuJ1) | Covnce | MMS-435p | 1:2000 dulution |
| GFAP antibody | Millipore | AB5804 | 1:1000 dilution |
| DAPI | Molecular Probes | D1306 | 1:2000 dilution |
| ZIKV antibody | World Reference Collection for Emerging Viruses and Arboviruses at the University of Texas Medical Branch | 1:2000 dilution | |
| Fluoromount G | SouthernBiotech | 0100-01 | |
| CO2 incubator | Thermo Forma | Model# 3110 | |
| Centrifuge | Thermo fisher Scientific | 75004221 | |
| Biological safety cabinet | Forma scientific Claas II A/B3 | Model# 1284 | |
| Freezer (-80 °C) | Forma scientific,Inc | model# 8516 | |
| Microscope(phase contrast image) | Hp 2230 workstation | Product#NOE25us#ABA | |
| Microscope (epifluorescent image) | Nikon Eclipse 80i | ||
| Confocal Microscope | Nikon TE2000-E microscope with C1si confocal system |
References
- Centers for Disease Control and Prevention. . All countries and territories with active Zika virus transmisson. , (2016).
- Brasil, P., et al. Zika Virus Infection in Pregnant Women in Rio de Janeiro. N Engl J Med. 375 (24), 2321-2334 (2016).
- Hills, S. L., et al. Transmission of Zika Virus Through Sexual Contact with Travelers to Areas of Ongoing Transmission – Continental United States, 2016. MMWR Morb Mortal Wkly Rep. 65 (8), 215-216 (2016).
- Centers for Disease Control and Prevention. . Facts about microcephaly. , (2016).
- Garcez, P. P., et al. Zika virus impairs growth in human neurospheres and brain organoids. Science. 352 (6287), 816-818 (2016).
- Li, C., et al. Zika Virus Disrupts Neural Progenitor Development and Leads to Microcephaly in Mice. Cell Stem Cell. 19 (5), 672 (2016).
- Tang, H., et al. Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Cell Stem Cell. 18 (5), 587-590 (2016).
- Wu, K. Y., et al. Vertical transmission of Zika virus targeting the radial glial cells affects cortex development of offspring mice. Cell Res. 26 (6), 645-654 (2016).
- Jensen, J. B., Parmar, M. Strengths and limitations of the neurosphere culture system. Mol Neurobiol. 34 (3), 153-161 (2006).
- Wu, P., et al. Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat. Nat Neurosci. 5 (12), 1271-1278 (2002).
- McGrath, E. L., et al. Differential Responses of Human Fetal Brain Neural Stem Cells to Zika Virus Infection. Stem Cell Reports. 8 (3), 715-727 (2017).
- Svendsen, C. N., et al. A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods. 85 (2), 141-152 (1998).
- Tarasenko, Y. I., Yu, Y., Jordan, P. M., Bottenstein, J., Wu, P. Effect of growth factors on proliferation and phenotypic differentiation of human fetal neural stem cells. J Neurosci Res. 78 (5), 625-636 (2004).
- Barrows, N. J., et al. A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection. Cell Host Microbe. 20 (2), 259-270 (2016).
- Jakel, R. J., Schneider, B. L., Svendsen, C. N. Using human neural stem cells to model neurological disease. Nat Rev Genet. 5 (2), 136-144 (2004).
- Lopez-Garcia, I., et al. Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro: identification of rifampicin as a neuroprotectant. Br J Pharmacol. , (2016).
- Mich, J. K., et al. Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain. Elife. 3, e02669 (2014).
