胚胎小鼠神经干细胞的分离与培养
Özet
在这里, 我们介绍了一种新的显微外科技术, 从 e13小鼠胚胎神经节隆起分离神经干细胞。
Abstract
神经干细胞是多能的, 可以产生中枢神经系统 (cns) 的三种主要细胞类型。nsc 的体外培养和扩开发为神经科学家研究神经元和胶质细胞的功能及其相互作用提供了合适的细胞来源。有几种技术被报道用于从成人或胚胎哺乳动物大脑中分离神经干细胞。在从胚胎中枢神经系统不同区域分离 nsc 的显微外科手术中, 减少对脑细胞的损害, 以获得最高比例的活干细胞和可膨胀干细胞是非常重要的。在从小鼠胚胎大脑中分离这些细胞时, 一种可能的减压技术是缩短手术时间。在这里, 我们展示了一种技术, 快速分离这些细胞从 e13小鼠胚胎神经节隆起。手术程序包括从子宫中取出 e13 小鼠胚胎, 用弯曲的针尖切割胚胎的额叶, 从头骨中取出大脑, 对分离的大脑进行显微解剖, 以获得神经节隆起,在 nsc 培养基中分离收获的组织以获得单个细胞悬浮液, 最后在悬浮培养中电镀细胞以产生神经球。
Introduction
神经干细胞 (nsc) 位于成人和胚胎大脑的不同区域, 它们有产生不同类型神经元和胶质细胞1的倾向。成年哺乳动物大脑2的下心室区和胚胎脑的神经节隆起是 nsc 丰富的区域3。在发育中的大脑中, 神经节隆起提供了大部分的皮质间质, 特别是 gabaep 的间质 3。还有一些从胚胎干细胞 (esc) 或诱导多能干细胞 (ipsc) 生成神经干细胞的侵入性较小的方法, 可以减少对动物使用的需求。尽管从 esc 或 ipsc 生成 nsc 是可能的4,5, 它有一些优点和缺点相比, 从成人或胚胎大脑 6,7的神经干细胞分离, 8。诱导 esc 和 ipsc 向神经元表型分化的方案总是耗时和成本消耗的, 成功率 (70-80% nestin 阳性细胞)5低于直接从动物大脑中分离出的 nsc (99% 以上的 nestin 阳性细胞)9。此外, 干细胞在几个通道10,11后失去其遗传稳定性和分化倾向。尽管还有其他关于将体细胞直接转化为 nsc 的新报告, 但这些细胞是基因工程的, 在每个实验室12中都不易获得。因此, 从动物大脑中分离神经干细胞的需求仍然很大;通过改进手术技术, 可以减少动物的使用量。通过缩短手术时间和改进技术, 可以使细胞远离损伤, 并从每只动物身上获得最高的 nsc 率。
在这里, 我们介绍了一种简化和可重复的技术, 从小鼠 e13胚胎大脑中分离神经干细胞。
Protocol
所有关于动物的手术和程序都得到了伊朗德黑兰罗扬研究所动物伦理委员会的批准。 1. 手术器械、洗涤缓冲器 (hpes-mem)、细胞培养培养基和细胞培养板的制备 根据常规灭菌指南, 通过高压灭菌, 对手术工具 (剪刀、手术刀刀片手柄和钳子) 进行消毒。 准备足够数量的 hepes-mem 缓冲液 (约100毫升对于每个怀孕的小鼠来说就足够了)。在 hpees-mem 缓冲液中加入高浓度的?…
Representative Results
微解剖, 细胞分离和神经球培养.在这里, 我们提出了一种快速有效的方法, 为小鼠 e13脑显微手术和神经干细胞的分离。本文表明, 通过额部 fontanelle 的精细破裂, 可以将整个大脑从 e13 小鼠胚胎头骨中取出。有了这种方法, 大脑承受的伤害就会更小, 从大脑不同部位分离出细胞是可能的。在 bfgf 和 egf 存在的情况下, 收获的细胞可以在 nsc 培养基中?…
Discussion
使用合适的神经干细胞来源对神经科学家来说非常重要。神经干细胞可以从胚胎大脑的不同区域获得, 它们可以产生特定类型的神经元和胶质细胞。诱导神经干细胞分化的方法有多种, 以诱导它们产生成熟的神经元和胶质细胞。有大量报道表明, 在特定的培养条件和营养因子群中, 它们可以在体外产生神经元14、少突胶质细胞15、16…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
这项工作得到了罗扬研究所的支持。
Materials
| 15 mL tubes | Falcon | 352097 | |
| 50 mL tubes | Falcon | 352235 | |
| Adson Forceps, 12 cm, Straight | WPI | 14226 | |
| B27 | Gibco | 17504-44 | |
| bFGF | Sigma | F0291 | |
| bovine serum albumin (BSA) | Sigma | A2153 | |
| dish 10cm | Falcon | 353003 | |
| Dressing Forceps, 12.5 cm, Straight | WPI | 15908 | |
| Dumont Tweezers, 11 cm, Straight | WPI | 500342 | |
| EGF | Sigma | E9644 | |
| Ethanol | Merck | 100983 | |
| Glutamate | Sigma | G3291 | |
| Glutamax | Gibco | 35050061 | |
| goat anti rabbit FITC conjugated secondary antibody | Sigma | AP307F | |
| goat serum | Sigma | G9023 | |
| Heparin | Sigma | h3149 | |
| HEPES | Sigma | 83264 | |
| HEPES | Sigma | 90909C | |
| insulinsyringe with 25-27 gauge Needle | SUPA medical | A1SNL127 | |
| laminin | sigma | L2020 | |
| MEM | Sigma | M2279 | |
| N2 supplement | Gibco | 17502048 | |
| NB medium | Gibco | 21103-31 | |
| Non-essential amino acid (NEAA) | Gibco | 11140050 | |
| PBS without Ca and Mg | Gibco | 20012050 | |
| Penicilin/ Streptomycin | Gibco | 5140122 | |
| Poly-L-ornithine | Sigma | P4957 | |
| rabbit anti mouse beta tubulin-III antibody | Sigma | T2200 | |
| rabbit anti mouse GFAP antibody | Sigma | G4546 | |
| rabbit anti mouse Nestin antibody | Sigma | N5413 | |
| Scalpel Handle #3 | WPI | 500236 | |
| Scissors curve | WPI | 14396 | |
| Scissors sharp straight | WPI | 14192 | |
| Soybean trypsin inhibitor | Roche | 10109886001 | |
| Tissue culture flasks, T25 | BD | 353014 | |
| Tissue culture flasks, T75 | BD | 353024 | |
| Tween 20 | Sigma | P1379 | |
| Vannas Scissors, 8 cm, Straight | WPI | 14003 | |
| β-mercaptoethanol | Sigma | M6250 |
Referanslar
- Navarro Quiroz, E., et al. Cell Signaling in Neuronal Stem Cells. Cells. 7 (7), (2018).
- Choi, C. I., et al. The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation. Scientific Reports. 8 (1), 9360 (2018).
- Li, H., et al. Isolation of a novel rat neural progenitor clone that expresses Dlx family transcription factors and gives rise to functional GABAergic neurons in culture. Developmental Neurobiology. 72 (6), 805-820 (2012).
- Liu, Y., et al. Derivation of phenotypically diverse neural culture from hESC by combining adherent and dissociation methods. Journal of Neuroscience Methods. , (2018).
- Dhara, S. K., et al. Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures. Differentiation. 76 (5), 454-464 (2008).
- Aligholi, H., Hassanzadeh, G., Gorji, A., Azari, H. A Novel Biopsy Method for Isolating Neural Stem Cells from the Subventricular Zone of the Adult Rat Brain for Autologous Transplantation in CNS Injuries. Methods in Molecular Biology. 1462, 711-731 (2016).
- Azari, H., Sharififar, S., Rahman, M., Ansari, S., Reynolds, B. A. Establishing embryonic mouse neural stem cell culture using the neurosphere assay. Journal of Visualized Experiments. (47), (2011).
- Ferrari, D., Binda, E., De Filippis, L., Vescovi, A. L. Isolation of neural stem cells from neural tissues using the neurosphere technique. Current Protocols in Stem Cell Biology. , (2010).
- Guo, W., Patzlaff, N. E., Jobe, E. M., Zhao, X. Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse. Nature Protocols. 7 (2012), (2005).
- Liu, P., et al. Passage Number is a Major Contributor to Genomic Structural Variations in Mouse iPSCs. Stem Cells and Development. 32 (10), 2657-2667 (2014).
- Diaferia, G. R., et al. Systematic Chromosomal Analysis of Cultured Mouse Neural Stem Cell Lines. Stem Cells and Development. 20 (8), 1411-1423 (2011).
- Hemmer, K., et al. Induced Neural Stem Cells Achieve Long-Term Survival and Functional Integration in the Adult Mouse Brain. Stem Cell Reports. 3 (3), 423-431 (2014).
- Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types. Journal of Visualized Experiments. (94), 52241 (2014).
- Zhang, Z., Wang, Z., Rui, W., Wu, S. Magnesium lithospermate B promotes proliferation and differentiation of neural stem cells in vitro and enhances neurogenesis in vivo. Tissue and Cell. 53, 8-14 (2018).
- Zilkha-Falb, R., Gurevich, M., Hanael, E., Achiron, A. Prickle1 as positive regulator of oligodendrocyte differentiation. Nörobilim. 364, 107-121 (2017).
- Wang, L., et al. Oligodendrocyte differentiation from human neural stem cells: A novel role for c-Src. Neurochemistry International. 120, 21-32 (2018).
- Palanisamy, A., et al. Oxytocin alters cell fate selection of rat neural progenitor cells in vitro. PLoS ONE. 13 (1), e0191160 (2018).
- Llewellyn-Smith, I. J., Basbaum, A. I., Braz, J. M. Long-term, dynamic synaptic reorganization after GABAergic precursor cell transplantation into adult mouse spinal cord. Journal of Comparative Neurology. 526 (3), 480-495 (2018).
- Precious, S. V., et al. FoxP1 marks medium spiny neurons from precursors to maturity and is required for their differentiation. Experimental Neurology. 282, 9-18 (2016).
Bu Makaleden Alıntı Yapın
Homayouni Moghadam, F., Sadeghi-Zadeh, M., Alizadeh-Shoorjestan, B., Dehghani-Varnamkhasti, R., Narimani, S., Darabi, L., Kiani Esfahani, A., Nasr Esfahani, M. H. Isolation and Culture of Embryonic Mouse Neural Stem Cells. J. Vis. Exp. (141), e58874, doi:10.3791/58874 (2018).