Summary

Выделение и культуры клеток нервного гребня из эмбриональных мышей нервной трубки

Published: June 02, 2012
doi:

Summary

Выделение эмбриональных нервного гребня из нервной трубки облегчает использование<em> В пробирке</em> Методы изучения миграции, самообновления и multipotency из нервного гребня.

Abstract

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone.

NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro.

To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23.

The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Protocol

1. Подготовка пластин Использование стерильных во все времена. Подготовить фибронектин (FN) путем разбавления 100 мкл плазмы человека запас FN в конечном объеме 3,3 мл в PBS Дульбекко (DPBS). Конечная концентрация 30 мкг / мл, и это можно хранить при температуре 4 ° С в течение 1 недели. …

Discussion

Особое внимание следует обратить на стадии развития эмбриона, чтобы обеспечить успех этого подхода. Подсчет сомитов ранних эмбрионов мыши имеет решающее значение как для стадии эмбриона в соответствие мусора и правильно определить регионы нервной трубки для изоляции. Изменение одно?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Мы хотели бы поблагодарить Марка Возняка для видео-помощь. Мы также хотели бы признать, Шон Моррисон в Техасском Университете в оригинальный протокол для культивирования клеток крысы NC. Работа выполнена при поддержке Университета Вандербильта Медицинского центра академической поддержки программ и грантов от NIH (HD36720 и HD036720-11S109) и АНА 11GRNT7690040 в PAL, predoctoral стипендии от AHA (0615209B) и NIH (NS065604), чтобы Движение неприсоединения, а также ERP было поддерживается обучение NIH грант T32HD007502.

Materials

Reagent Company Catalogue number Comments
DMEM (low glucose) Gibco/Invitrogen 11885  
Neurobasal Medium Gibco 21103  
BSA Sigma A3912-10G  
dPBS Gibco 14190-144  
IGF1 BD Biosciences 354037 Store in 50 μg/mL aliquots at -20°C.
bFGF BD Biosciences 354060 Store in 25 μg/mL aliquots at -20°C.
Fibronectin Gibco 33016-015 Stored in 1mg/mL aliquots at -20°C.
Retinoic acid Sigma R2625 Store in 35 μg/ml aliquots after reconstituting in ethanol at -20°C.
2-mercaptoethanol Sigma D-5637  
N2 supplement Gibco 17502-048  
B27 supplement Gibco 17504-044  
Steriflip 0.22 μm filters Millipore SCGP00525  
Penicillin-streptomycin Invitrogen 15140122  
0.20 μm filters Corning 431219  
Syringes (for filtration) BD Biosciences 301604  
Four well plates Thermo Fisher Scientific 176740  
Collagenase/Dispase Roche 269 638 Activity varies by batch. Store in 100 mg/mL aliquots at -20°C.
Insulin needles
(29½ gage)
Becton Dickson 309306  
Hypoxia Chamber Billups-Rothenberg    
Oxygen Analyzer Billups-Rothenberg    
Forceps #5 Fine Science Tools   For removing uterus and decidua.
Trypsin-EDTA (0.25%) Gibco 25200  

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Cite This Article
Pfaltzgraff, E. R., Mundell, N. A., Labosky, P. A. Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube. J. Vis. Exp. (64), e4134, doi:10.3791/4134 (2012).

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