We would like to acknowledge Marc Wozniak for video assistance. We would also like to acknowledge Sean Morrison at UT Southwestern for the original protocol for culturing rat NC cells. This work was supported Vanderbilt University Medical Center Academic Program Support and by grants from the NIH (HD36720 and HD036720-11S109) and the AHA 11GRNT7690040 to PAL, predoctoral fellowships from the AHA (0615209B) and NIH (NS065604) to NAM, and ERP was supported by an NIH training grant T32HD007502.

1. Preparing Plates
<ol>
 <li> Use sterile technique at all times. </li>
 <li> Prepare fibronectin (FN) by diluting 100 &mu;L of human plasma FN stock into a final volume of 3.3 mL in Dulbecco's PBS (dPBS). Final concentration is 30 &mu;g/mL and this can be stored at 4 &deg;C for 1 week. </li>
 <li> 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). </li>
 <li> Remove FN solution and allow plates to dry. Gently rinse wells with 500 &mu;L DMEM, remove DMEM, and add 500 &mu;L of Self-Renewal (SR) medium (see below). Incubate at 37 &deg;C in a humidified incubator containing 5 percent CO2. Complete this approximately one hour before dissection so that the plates will be dry before use. </li>
</ol>
2. Preparing SR Medium
<ol>
 <li> For the culture of vagal and trunk NC from ten embryos (approximately one litter, depending on genetic background of the mouse line), prepare 25 mL of SR medium. Combine 12.5 mL low glucose DMEM, 7.5 mL Neurobasal Medium, 25 &mu;L retinoic acid (117 &mu;M final concentration), and 25 &mu;L 2-mercaptoethanol (50 mM final concentration). Mix well.</li>
 <li> Add 3.75 mL Chick Embryo Extract, 250 &mu;L N2 salt supplement, 500 &mu;L B27 supplement and 250 &mu;L penicillin-streptomycin (1% final concentration). Filter the medium through a 0.22 &mu;m filter.</li>
 <li> Add 10 &mu;L sterile IGF1 (20 &mu;g/mL final concentration) and 20 &mu;L sterile bFGF (20 &mu;g/mL final concentration). Mix by inverting. Store at 4 &deg;C. </li>
</ol>
3. Preparing Wash Medium
<ol>
 <li> For approximately 10 embryos prepare 50 mL of wash medium. Combine 50 mg BSA with 35 mL low glucose DMEM, 15 mL Neurobasal Medium, and 500 &mu;L penicillin-streptomycin (1% final concentration). Sterile filter with a 0.22 &mu;m filter. </li>
</ol>
4. Preparing Collagenase/dispase
<ol>
 <li> Add 50 &mu;L of 100 mg/mL collagenase/dispase to 5 mL dPBS. Mix well.</li>
 <li> Syringe filter with a 0.2 &mu;m filter and add 1.5 mL into each of three wells of a twelve well plate. Pipette approximately 1 mL wash medium in the remaining wells. Store entire plate on ice until ready to digest dissected tissue. </li>
 <li> Cut tips of a p20 and p1000 pipette filter tip with a sterile razor blade. Cut just below the beveled edge of the tip. The cut off p1000 will be used to transfer the whole embryo while the p20 will be used to transfer pieces of isolated tissue. </li>
</ol>
5. Isolating Vagal and Trunk Neural Tube from 9.5 dpc Embryos
<ol>
 <li> For timed pregnancies, dams with a vaginal plug are considered 0.5 dpc at noon the morning the plug is observed. Sacrifice and remove the uterus at 9.5 dpc. </li>
 <li> Remove the decidua from uterus and gently remove the embryo from the decidua. Once experienced with this protocol, isolate 3-4 9.5 dpc embryos at a time in sterile dPBS. Use sterile technique and sterilized dissection instruments. Instruments can be sterilized by autoclaving, heat sterilization or by incubation in ethanol. </li>
 <li> For anterior vagal NC: using insulin needles, cut the neural tube at the mid otic placode. Cut again at the posterior edge of the 4th somite. Trim tissue ventral to the neural tube to remove the pharyngeal arches and heart. </li>
 <li> For trunk NC: using insulin needles, remove the portion of the neural tube between somites 16-22 (or the last somite if embryos are developmentally earlier than the 22 somite stage). </li>
 <li> Keep the yolk sac and any remaining embryonic tissue for genotyping.</li>
</ol>
6. Removal of Non-neural Ectoderm and Mesoderm
<ol>
 <li> Place neural tube containing segments into collagenase/dispase at room temperature for 10 minutes. Immediately wash in wash medium. </li>
 <li> Return tissue to sterile dPBS. Using sterile insulin needles, gently remove the non-neural ectoderm from the tissue and separate the somites away from the neural tube. The last remaining parts of the mesoderm from somite tissue can be removed by triturating gently using a cut-down p20 tip. Be careful to not damage the neural tube. Observe this closely during trituration. </li>
 <li> Place the isolated neural tube through a second and third 30 second wash of wash medium.</li>
 <li> Wash once in SR medium, and place the isolated neural tube into the center of a FN-coated well that was prepared previously (Step 1.3). Humidify the hypoxia chamber with a dish of sterile water. Place the dish into the hypoxia chamber and flush the chamber with mixed gas to 3% O2 (use a tank containing a mixture of 1% O2, 6% CO2, 93% N2). </li>
 <li> Always handle the chamber extremely carefully to ensure that explants are undisturbed and remain in the center of the wells. </li>
 <li> Incubate at 37 &deg;C. Summary of steps 5-6 is shown in Figure 1.</li>
</ol>
7. Removal of Neural Tube
<ol>
 <li> After 24 hours of incubation, remove the neural tube by gently teasing the edge of the neural tube away from the migrating cells using a sterile insulin needle. Remove and discard the neural tube from the medium using a sterile p20 cut as in step 6.2 and replace the medium with fresh SR medium (Figure 2). This is most easily done using an inverted microscope. </li>
</ol>
8. Representative Results
Following 24 hours of incubation at 37 &deg;C in hypoxic conditions, NC cells have migrated away from the neural tube in a nearly pure population (Figure 3a). Sometimes, less than ideal cultures will not yield robust outgrowths. For example, it is possible that after 24 hours the neural tube will have curled up upon itself and the NC will not migrate away from the neural tube (Figure 3b). Occasionally the neural tube will not attach to the fibronectin coated plates. 
In our experience, sub-optimal NC migration or problems with attachment of the neural tube can be adversely affected by normoxia conditions or concentration of the fibronectin, respectively. Enzymatic activity of the collagenase/dispase varies slightly by batch and digestion time must be adjusted appropriately, however, do not digest the tissue longer than fifteen minutes. Overdigestion of the neural tube containing tissue in collagenase/dispase will also result in deficient outgrowths. If somite tissue is not easily removed from the neural tube after incubation in collagenase/dispase, the neural tube can be incubated for longer than ten minutes. Sometimes the neural tube will not attach to the substrate. If this is the case, double-check the fibronectin concentration and the hypoxia conditions. 
While normoxic conditions can be used to culture wild-type NC, hypoxic conditions more closely mimic the in vivo environment29,30. In our experience, hypoxic conditions became critical when culturing mutant NC. For example, when Foxd3 mutant NC were cultured in normoxic conditions, trunk NC had a greatly reduced cell outgrowth compared to controls. This disparity in outgrowth size was removed when the explants were cultured in hypoxic conditions (Figure 4). Furthermore, when wild-type neural tube explants were cultured in normoxia, the number of caspase-positive cells was greater then that of similar explants cultured in hypoxia (data not shown). By maintaining all NC culture in hypoxia, comparisons can more easily be made between dynamics of control and mutant cultures.
<img src="/files/ftp_upload/4134/4134fig1.jpg" alt="Figure 1" /> 
Figure 1. Overall schematic of NC isolation. A) Dissect regions of interest from the embryo. B) Digest neural tube in collagenase/dispase for ten minutes (do not exceed fifteen minutes). C) Wash in wash medium. D) Dissect away the non-neural ectoderm and mesoderm. E-F) Wash twice in wash medium. G) Plate in self renewal medium. Incubate at 37 &deg;C in 3% O2 hypoxic conditions.
<img src="/files/ftp_upload/4134/4134fig2.jpg" alt="Figure 2" /> 
Figure 2. Stepwise removal of neural tube from explant. The neural tube must be removed after 24-48 hours to prevent contamination with non-NC cells. A) Notice the boundary between the neural tube and the NC outgrowth (solid line). B) Cut along the edge of the neural tube with an insulin needle. C) Discard the neural tube and replace medium with fresh self renewal medium. Dashed line indicates extent of the outgrowth. Abbreviation: NT, neural tube.
<img src="/files/ftp_upload/4134/4134fig3.jpg" alt="Figure 3" /> 
Figure 3. Examples of representative results. A) Typical explant outgrowth after 24 hours incubation in self renewal medium in hypoxic conditions (dashed line indicates extent of the outgrowth). B) Magnified view of NC outgrowth after 48 hours in culture. C) Less ideal culture with low outgrowth yield. A and C were cultured under the same conditions. Images demonstrate the natural range in culture robustness. This can be affected by efficiency of neural tube isolation, concentration of FN, hypoxic conditions, and time in collagenase/dispase digestions. 
<img src="/files/ftp_upload/4134/4134fig4.jpg" alt="Figure 4" /> 
Figure 4. In vitro analyses of NC explant cultures in normoxia versus hypoxia. Control (wild type) NC cells migrated from neural tube explants after 48 hours in normoxic culture conditions. In contrast, Foxd3 mutant NC had greatly reduced cell outgrowths in normoxia (red outlines mark edges of the NC outgrowths). When comparable explants were grown under hypoxic conditions, Foxd3 mutant NC explants grew comparably to controls, allowing subsequent analyses. Note, this behavior correlated well with the behavior of Foxd3 mutant NC in vivo.

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We have nothing to disclose.

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 more than 25 somites, the NC is further advanced in developmental time, and NC outgrowths will be less robust in culture. Staging embryos based on somite number facilitates precision in experiments when developmental stage of the embryo, and therefore NC, can influence the outcome of the experiments. In a 9.5 dpc embryo, the vagal NC migrates from the dorsal neural tube in defined anterior-posterior limits: from the mid-otic placode to somite 7. Similarly, trunk NC migrates from the neural tube at levels somite 8 to somite 24. To isolate distinct populations of NC progenitors as described in this protocol, discrete sub-regions within each of those domains are isolated. The region of the neural tube used will vary based upon the design of the experiment and the anterior-posterior region of the NC being studied.
The culture conditions described above, including the medium and incubation conditions, are specifically adapted for culture of murine NC. Similar SR medium was first used for the culture of rat NC11. In our hands, this rat medium, with the addition of Neurobasal Medium, produces a robust outgrowth of murine NC progenitors. Furthermore, unlike previous work culturing avian NC cells, a feeder layer is unnecessary for culture of the mammalian NC using this method11,13,21,22. While there are numerous protocols detailing both avian and mammalian NC culture in normoxic conditions9,22, we23 (Figure 4), along with others in the field20, have found that culturing murine NC cells in hypoxic conditions as described here greatly aids survival and self-renewal, presumably because hypoxia more closely mimics physiological oxygen levels within the embryo20,29,30.
Evaluation of the expression of molecular markers greatly facilitates the identification of NC cells and their differentiated derivatives. These include expression of Foxd3, p75, and Sox10 for NC stem cells. Markers of differentiated NC include smooth muscle alpha actin (SMA) for myofibroblasts, glial fibrillary acidic protein (GFAP) for glia, microphthalmia-associated transcription factor (MITF) for melanocytes, and beta-III tubulin, protein gene product 9.5 (PGP9.5), and peripherin for neurons. These markers can be used to determine explant efficiency and differentiation of NC progenitors.
Once this technique is mastered, cultured NC can be used in a variety of assays including quantification of proliferation, cell death, migration dynamics, and/or differentiation status. NC can also be clonally cultured to investigate self-renewal or multipotency of individual NC progenitors23. After the neural tube is removed from the cultures, dissociate the NC cells in 100 &mu;l Trypsin-EDTA (0.25%) for exactly 5 minutes at 37 &deg;C. Quench Trypsin-EDTA in excess wash medium (8-10 mLs) by adding 800 &mu;l per well and transferring to individual 15 mL tubes containing wash medium. Gently centrifuge cells at 150 x g for 3 minutes, remove supernatant and resuspend cell pellet in 1 mL SR medium. Count cells using a hemocytometer and plate them at a density of 25 cells/cm2. These colonies can be serially passaged to assay self-renewal. To assay multipotency, maintain the NC cells at clonal density in SR medium for 6 days and then switch to differentiation medium (10 ng/mL bFGF and 1% chick embryo extract). Culture the cells in differentiation medium under hypoxia for 8 days before analyzing colony composition with molecular markers as above11. 
In conclusion, this method for isolating NC cells from mouse embryos produces a feeder free adherent culture of premigratory NC cells without the use of FACS. Analysis of experiments using these cells can easily be quantified. While this technique is straightforward, its applications for the study of NC characteristics of migration, self-renewal, and differentiation are vast. Furthermore, isolating NC from genetically modified mouse embryos allows for the direct study of particular genes and pathways in the context of NC migration, survival, and/or differentiation.

<table border="1">
<tbody>
 <tr>
 <td>Reagent </td>
 <td>Company </td>
 <td>Catalogue number </td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>DMEM (low glucose)</td>
 <td>Gibco/Invitrogen</td>
 <td>11885</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Neurobasal Medium</td>
 <td>Gibco</td>
 <td>21103</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A3912-10G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dPBS</td>
 <td>Gibco</td>
 <td>14190-144</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>IGF1</td>
 <td>BD Biosciences</td>
 <td>354037</td>
 <td>Store in 50 &mu;g/mL aliquots at -20&deg;C. </td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>BD Biosciences</td>
 <td>354060</td>
 <td>Store in 25 &mu;g/mL aliquots at -20&deg;C. </td>
 </tr>
 <tr>
 <td>Fibronectin</td>
 <td>Gibco</td>
 <td>33016-015</td>
 <td>Stored in 1mg/mL aliquots at -20&deg;C. </td>
 </tr>
 <tr>
 <td>Retinoic acid</td>
 <td>Sigma</td>
 <td>R2625</td>
 <td>Store in 35 &mu;g/ml aliquots after reconstituting in ethanol at -20&deg;C. </td>
 </tr>
 <tr>
 <td>2-mercaptoethanol </td>
 <td>Sigma</td>
 <td>D-5637</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>N2 supplement</td>
 <td>Gibco</td>
 <td>17502-048</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B27 supplement</td>
 <td>Gibco</td>
 <td>17504-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Steriflip 0.22 &mu;m filters</td>
 <td>Millipore</td>
 <td>SCGP00525</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin </td>
 <td>Invitrogen</td>
 <td>15140122 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.20 &mu;m filters</td>
 <td>Corning</td>
 <td>431219</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Syringes (for filtration)</td>
 <td>BD Biosciences</td>
 <td>301604</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Four well plates</td>
 <td>Thermo Fisher Scientific</td>
 <td>176740</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase/Dispase</td>
 <td>Roche</td>
 <td>269 638</td>
 <td>Activity varies by batch. Store in 100 mg/mL aliquots at -20&deg;C.</td>
 </tr>
 <tr>
 <td>Insulin needles 
 (29&frac12; gage)</td>
 <td>Becton Dickson</td>
 <td>309306</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hypoxia Chamber</td>
 <td>Billups-Rothenberg</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oxygen Analyzer</td>
 <td>Billups-Rothenberg</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Forceps #5</td>
 <td>Fine Science Tools</td>
 <td>&nbsp;</td>
 <td>For removing uterus and decidua.</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA (0.25%) </td>
 <td>Gibco</td>
 <td>25200</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

isolation and culture of neural crest cells from embryonic murine neural tube

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.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

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

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.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

isolation-culture-neural-crest-cells-from-embryonic-murine-neural

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.1K Views.  Vanderbilt University Medical Center. 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.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...