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 ...

<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 ...

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Research

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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 ...

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 ...

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 ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...