Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Summary

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

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 organs^4-6. 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 tube^7-9. 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 embryo^10-13. 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 progenitors^11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties^13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors^11,13,14,17. 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 NC^11,13. 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 NC^2,24-28.

Protocol

1. Preparing Plates

1. Use sterile technique at all times.
2. Prepare fibronectin (FN) by diluting 100 μL of human plasma FN stock into a final volume of 3.3 mL in Dulbecco's PBS (dPBS). Final concentration is 30 μg/mL and this can be stored at 4 °C for 1 week.
3. Cover bottom of each well of a sterile tissue culture four well plate with FN solution and let sit for 15 minutes. Make sure the entire surface is covered. Make media during this time (steps 2 and 3).
4. Remove FN solution and allow plates to dry. Gently rinse wells with 500 μL DMEM, remove DMEM, and add 500 μL of Self-Renewal (SR) medium (see below). Incubate a....

Discussion

Careful attention should be paid to the developmental stage of embryo to ensure the success of this approach. Counting somites of early mouse embryos is critical both for stage matching embryos within a litter and determining the correct regions of neural tube for isolation. A variation of one or two somites between embryos is within a reasonable range of developmental timing, depending upon the resolution of the experiment conducted. An embryo between 9 and 9.5 dpc will have between 17 and 25 somites. If the embryo has .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM (low glucose)	GIBCO, by Life Technologies	11885
Neurobasal Medium	GIBCO, by Life Technologies	21103
BSA	Sigma-Aldrich	A3912-10G
dPBS	GIBCO, by Life Technologies	14190-144
IGF1	BD Biosciences	354037	Store in 50 μg/mL aliquots
bFGF	BD Biosciences	354060	Store in 25 μg/mL aliquots
Fibronectin	GIBCO, by Life Technologies	33016-015	Stored in 1mg/mL aliquots
Retinoic Acid	Sigma-Aldrich	R2625	Put into solution with ethanol, make 35 μg/ml aliquots
2-mercapt–thanol	Sigma-Aldrich	D-5637
N2 supplement	GIBCO, by Life Technologies	17502-048
B27 supplement	GIBCO, by Life Technologies	17504-044
Steriflip 0.22 μm filters	EMD Millipore	SCGP00525
Penicillin/streptomycin	Invitrogen	15140122
0.20 μm filters	Corning	431219
Syringes (for filtration)	BD Biosciences	301604
Four well plates	Thermo Fisher Scientific, Inc.	176740
Collagenase/Dispase	Roche Group	269 638	Activity varies by batch. Stored in 100 mg/mL aliquots.
Insulin needles (29 ½ gage)	BD Biosciences	309306
Hypoxia Chamber	Billups-Rothenberg, Inc.
Oxygen Analyzer	Billups-Rothenberg, Inc.
Forceps #5	Fine Science Tools		For removing uterus and decidua.