Nicole Stancel, Ph.D., ELS, of Scientific Publications at the Texas Heart Institute, provided editorial support. We also thank the following funding sources: the American Heart Association&#39;s National Center Scientist Development Grant (14SDG19840000 to J. Wang), the 2014 Lawrence Research Award from the Rolanette and Berdon Lawrence Bone Disease Program of Texas (to J. Wang), and the National Institutes of Health (DE026561 and DE025873 to J. Wang, DE016320 and DE019650 to R. Maxson).

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+neural+crest+stem+cell-derived+dopaminergic+neuronal+model+recapitulates+biochemical+abnormalities+in+GBA1+mutation+carriers.">A human neural crest stem cell-derived dopaminergic neuronal model recapitulates biochemical abnormalities in GBA1 mutation carriers.</a> Stem Cell Reports. 8 (3), 728-742 (2017).</li><li>Ishii, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stable+cranial+neural+crest+cell+line+from+mouse.">A stable cranial neural crest cell line from mouse.</a> Stem Cells and Development. 21 (17), 3069-3080 (2012).</li><li>Yumoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta-activated+kinase+1+(Tak1)+mediates+agonist-induced+Smad+activation+and+linker+region+phosphorylation+in+embryonic+craniofacial+neural+crest-derived+cells.">TGF-beta-activated kinase 1 (Tak1) mediates agonist-induced Smad activation and linker region phosphorylation in embryonic craniofacial neural crest-derived cells.</a> Journal of Biological Chemistry. 288 (19), 13467-13480 (2013).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yap+and+Taz+play+a+crucial+role+in+neural+crest-derived+craniofacial+development.">Yap and Taz play a crucial role in neural crest-derived craniofacial development.</a> Development. 143 (3), 504-515 (2016).</li><li>Heallen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippo+pathway+inhibits+Wnt+signaling+to+restrain+cardiomyocyte+proliferation+and+heart+size.">Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size.</a> Science. 332 (6028), 458-461 (2011).</li><li>Wang, J., Martin, J. 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The authors have nothing to disclose.

The NCC is a versatile and key contributor to different tissues and organs during embryonic morphogenesis.The O9-1 cell line maintains its potential to differentiate into many different cell types and mimics the in vivo characteristics of NCCs, making it a useful in vitro tool for studying gene function and molecular regulation in NCCs. The different status of O9-1 cells may correspond to different neural crest progeny in vivo, depending on the culture conditions of O9-1 cells. O9-1 cells can b...

An erratum was issued for: <a href="https://www.jove.com/video/58346/culturing-and-manipulation-of-o9-1-neural-crest-cells">Culturing and Manipulation of O9-1 Neural Crest Cells</a>. The Protocol section was updated.
Step 2.1 was updated from:
Prepare basal media for O9-1 cell culture by adding the following in DMEM (final concentrations are indicated): 15% FBS, 0.1 mM minimum essential media (MEM) nonessential amino acids, 1 mM sodium pyruvate, 55 mM beta-mercaptoethanol, 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM L-glutamine, 103 units/mL leukemia inhibitory factor (LIF; added immediately before use, do not add to stock bottle), and 25 ng/mL fibroblast growth factor-basic (bFGF; added immediately before use, do not add to stock bottle).
to:
Prepare basal media for O9-1 cell culture by adding the following in DMEM (final concentrations are indicated): 15% FBS, 0.1 mM minimum essential media (MEM) nonessential amino acids, 1 mM sodium pyruvate, 55 &#181;M beta-mercaptoethanol, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 2 mM L-glutamine, 103 units/mL leukemia inhibitory factor (LIF; added immediately before use, do not add to stock bottle), and 25 ng/mL fibroblast growth factor-basic (bFGF; added immediately before use, do not add to stock bottle).
Step 5.1.1 was updated from:
To prepare osteogenic differentiation media, dilute the following in alpha-MEM (final concentrations are indicated): 0.1 mM dexamethasone, 100 ng/mL bone morphogenetic protein 2 (BMP2), 50 &#181;g/mL ascorbic acid, 10 mM b-glycerophosphate, 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin.
to:
To prepare osteogenic differentiation media, dilute the following in alpha-MEM (final concentrations are indicated): 0.1 &#181;M dexamethasone, 100 ng/mL bone morphogenetic protein 2 (BMP2), 50 &#181;g/mL ascorbic acid, 10 mM b-glycerophosphate, 10% FBS, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin.
Step 5.2.1 was updated from:
To prepare chondrocyte differentiation media, dilute the following in alpha-MEM (final concentrations are indicated): 5% fetal calf serum (FCS), 1% insulin-transferrin-selenium (ITS), 100 U/mL penicillin, 100 mg/mL streptomycin, 10 ng/mL transforming growth factor beta (TGF-b3), 50 mg/mL ascorbic acid, 10 ng/mL BMP2, 0.1 mM dexamethasone, and 1 mM sodium pyruvate.
to:
To prepare chondrocyte differentiation media, dilute the following in alpha-MEM (final concentrations are indicated): 5% fetal calf serum (FCS), 1% insulin-transferrin-selenium (ITS), 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 10 ng/mL transforming growth factor beta (TGF-b3), 50 &#181;g/mL ascorbic acid, 10 ng/mL BMP2, 0.1 &#181;M dexamethasone, and 1 mM sodium pyruvate.
Step 5.4.1 was updated from:
To prepare glial cell differentiation media, dilute the following in DMEM/F12 (final concentrations are indicated): 1x B-27 supplement, 2 mM L-glutamine, 50 ng/mL BMP2, 100 U/mL penicillin, 100 mg/mL streptomycin, 50 ng/mL LIF, and 1% heat-inactivated FBS.
to:
To prepare glial cell differentiation media, dilute the following in DMEM/F12 (final concentrations are indicated): 1x B-27 supplement, 2 mM L-glutamine, 50 ng/mL BMP2, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 50 ng/mL LIF, and 1% heat-inactivated FBS.

An erratum was issued for: <a href="https://www.jove.com/video/58346/culturing-and-manipulation-of-o9-1-neural-crest-cells">Culturing and Manipulation of O9-1 Neural Crest Cells</a>. The Protocol section was updated.

Erratum: Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are multipotent stem-like cells with a remarkable migratory ability and transient existence during embryonic development. NCCs originate between the surface ectoderm and the neural tube and migrate to other parts of the embryo during embryonic development1. Based on their functional domains, NCCs can be classified into several different types, including cranial, trunk, vagal, sacral, and cardiac NCCs. In addition, NCCs can differentiate into multiple cell lineages, such as smooth muscle cells, bone cells, and neurons, and give rise to various tissues2,3. The development of NCCs is characterized by a complex series of morphogenetic events that are fine-tuned by various molecular signals. Given the complex regulation of NCCs and their important contributions to numerous structures, the dysregulation of NCC development can commonly lead to congenital birth defects, which account for nearly 30% of all human congenital birth defects. Abnormalities during the neural crest development can lead to cleft lip/palate, flawed nose formation, syndromes, defects such as a defective cardiac outflow tract, or even infant mortality1,4,5,6,7. Understanding the molecular mechanisms of NCC development is important for developing treatments for diseases caused by defects in NCC development. With the use of various in vitro and in vivo approaches8,9,10,11,12,13,14,15, considerable progress has been made in NCC research. In vivo, animal models, including chickens, amphibians, zebrafish, and mice, have been used to investigate NCCs1. Furthermore, human embryos have been used to study the process of NCC migration in early human embryo development16. In vitro, cell models for NCCs, such as human NCCs that originated from patient subcutaneous fat, have been used to investigate Parkinson&#39;s disease17. The O9-1 NCC line, which was originally derived from mass cultures of endogenous NCCs isolated from E8.5 mouse embryos18, is a powerful cell model for studying NCCs. Importantly, under non-differentiating culture conditions, O9-1 cells are multipotent stem-like NCCs. However, under varying culture conditions, O9-1 cells can be differentiated into distinguished cell types, such as smooth muscle cells, osteoblasts, chondrocytes, and glial cells18. Given these properties, O9-1 cells have been broadly used for NCC-related studies, such as investigating the molecular mechanism of cranial-facial defects19,20.
Here, detailed protocols are provided for maintaining O9-1 cells, differentiating O9-1 cells into different cell types, and manipulating O9-1 cells by performing gene loss-of-function studies with CRISPR-Cas9 genome editing and small interfering RNA (siRNA)-mediated knockdown technologies. As a representative example, the use of O9-1 cells to study Yap and Taz loss-of-function is described. Yap and Taz are the downstream effectors of the Hippo signaling pathway, which plays a critical role in the cell proliferation, differentiation, and apoptosis. The Hippo pathway has also been shown to be important in the development and homeostasis of several different tissues and organs, as well as in the pathogenesis of different diseases20,21,22,23,24,25,26,27,28. The core components of Hippo signaling include the tumor suppressor sterile 20-like kinases Mst1/2, WW domain-containing Salvador scaffold protein, and the large tumor suppressor homolog (Lats1/2) kinases. Hippo signaling inhibits Yap and Taz activity and promotes their degradation in the cytoplasm. Without repression from Hippo, Yap and Taz can translocate into nuclei and function as transcriptional co-activators. We recently showed that specifically inactivating the Hippo signaling effectors Yap and Taz in mouse NCCs by using the Wnt1cre and Wnt1Cre2SOR drivers resulted in embryonic lethality at E10.5 with severe craniofacial defects20. We have also performed studies using O9-1 cells to investigate the role of Yap and Taz in NCCs. To study Yap and Taz function in NCC proliferation and differentiation, Yap and Taz knockdown cells were generated in O9-1 cells by using siRNA, and Yap knockout cells were generated by using CRISPR-Cas9 genome editing. The same gene loss-of-function strategies can be applied to different target genes in other pathways. In addition, gain-of-function studies and transfection assays can also be applied to O9-1 cells to study gene function and regulation. The protocols described here are intended to be used by investigators as guides for culturing O9-1 cells to maintain multipotent stemness, for differentiating O9-1 cells into other cell types under different culture conditions, and for studying gene function and the molecular mechanisms of NCCs.

<table><tbody><tr><td>Active STO feeder cells</td><td>ATCC</td><td>ATCC CRL-1503</td><td>Also available in mitomycin C-inactivated form, catalog # ATCC 56-X</td></tr><tr><td>O9-1 mouse cranial neural crest cell line</td><td>Millipore Sigma</td><td>SCC049</td><td></td></tr><tr><td>DMEM, high glucose, no glutamine</td><td>Gibco</td><td>11960-044</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Hyclone</td><td>SH30243.01</td><td></td></tr><tr><td>FBS (fetal bovine serum)</td><td>Millipore Sigma</td><td>ES-009-B</td><td></td></tr><tr><td>Penicillin - streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>L-glutamine 200 mM (100x)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>G1890</td><td></td></tr><tr><td>Trypsin-EDTA 0.25% in HBSS</td><td>Genesee Scientific</td><td>25-510</td><td></td></tr><tr><td>DPBS (Dulbecco&#039;s phosphate buffered saline) without calcium or magnesium</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>MEM non-essential amino acids (MEM NEAA) 100Xx</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Sodium pyruvate (100 mM)</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M-7522</td><td></td></tr><tr><td>ESGRO leukemia inhibitory factor (LIF) 10&lt;sup&gt;6&lt;/sup&gt; unit/mL</td><td>Millipore Sigma</td><td>ESG1106</td><td></td></tr><tr><td>Recombinant human fibroblast growth factor-basic (rhFGF-basic)</td><td>R&amp;amp;D Systems</td><td>233-FB-025</td><td></td></tr><tr><td>Mitomycin C</td><td>Roche</td><td>10107409001</td><td></td></tr><tr><td>Matrigel matrix</td><td>Corning</td><td>356234</td><td></td></tr><tr><td>DMSO (dimethylsulfoxide)</td><td>Millipore Sigma</td><td>MX1458-6</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Thermo Fisher Scientific</td><td>13778-075</td><td></td></tr><tr><td>Opti-MEM I (1x)</td><td>Gibco</td><td>31985-070</td><td></td></tr><tr><td>Minimum essential medium, alpha 1x with Earle&#039;s salts, ribonucleosides, deoxyribonucleosides, &amp;amp; L-glutamine</td><td>Corning</td><td>10-022-CV</td><td></td></tr><tr><td>ON-TARGETplus Wwtr1 siRNA</td><td>Dharmacon</td><td>L-041057</td><td></td></tr><tr><td>ON-TARGETplus Non-targeting Pool</td><td>Dharmacon</td><td>D-001810</td><td></td></tr><tr><td>ON-TARGETplus Yap1 siRNA</td><td>Dharmacon</td><td>L-046247</td><td></td></tr><tr><td>FCS (fetal calf serum)</td><td></td><td></td><td></td></tr><tr><td>ITS (insulin-transferrin-selenium)</td><td></td><td></td><td></td></tr><tr><td>TGF-b3</td><td></td><td></td><td></td></tr><tr><td>Ascorbic acid</td><td></td><td></td><td></td></tr><tr><td>BMP2 (bone morphogenetic protein 2)</td><td></td><td></td><td></td></tr><tr><td>Dexamethasone</td><td></td><td></td><td></td></tr><tr><td>B-27 supplement</td><td></td><td></td><td></td></tr></tbody></table>

culturing and manipulation of o9-1 neural crest cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

The goal of our knockdown and knockout experiments was to study the effects of Yap and Taz loss-of-function in O9-1 cells. Before the knockdown and knockout experiments, we have to make sure that prepare for basal media and culture O9-1 cells as described above (for example, basement membrane matrix needs to cover the whole plate as shown in Figure 1, and O9-1 cells recovered from liquid nitrogen as shown in Figure 2</st...

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Culturing and Manipulation of O9-1 Neural Crest Cells

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

culturing-and-manipulation-of-o9-1-neural-crest-cells

Research

JoVE Journal

Genetics

9.9K Views.  Baylor College of Medicine. De O9-1 cellijn is een zeer nuttig hulpmiddel voor het bestuderen van neurale kuifcellen in vitro. Het heeft een groot potentieel voor gebruik in een breed scala van toepassingen in verschillende gebruik van onderzoek. O9-1 cellen hebben duidelijke voordelen, zoals gemakkelijke toegang, en snel het verkrijgen van voldoende celnummers voor experimenten, waardoor het een krachtige methode gratis voor in vitro studies van neurale kuifcellen.Om te beginnen, bereid basale media voor O9-1 ce...

Video: Culturing and Manipulation of O9-1 Neural Crest Cells

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 ...

Neuroscience

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 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.

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 ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Developmental Biology

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest in studying the mechanisms regulating cell fate decisions and plasticity. Originating in the dorsal neuroepithelium, this cell population is transient and relatively rare in the developing embryo - making functional tests, genomic screens, and biochemistry assays challenging to perform in vivo. To overcome these limitations, several methods have been developed to model CNCC development in vitro. Neurosphere (NS) based culturing methods provide a complex microenvironment that recapitulates the developing anterior neuroepithelium in 3D. These systems allow the growth of many NS in the same plate to generate a large amount of CNCC, but the produced NS present a high variability in shape, size, and number of CNCC formed - making quantitative assays difficult to perform. This protocol outlines a reproducible method for generating NS from mouse embryonic stem cells (mESC) in a 96-well format. NS generated in 96-well plates produce cranial neural crest cells (CNCC), which can be further cultured. By controlling the number of starting cells, this approach reduces variability in the size and shape between NS and increases reproducibility across experiments. Finally, this culture system is adaptable to several applications and offers a higher degree of flexibility, making it highly customizable and suitable for multiplexing experimental conditions.

We present a three-dimensional (3D) in vitro differentiation protocol generating neurospheres of reproducible size to produce cranial neural crest cells from mouse embryonic stem cells. We show that this methodology reduces variability compared to previous protocols and how it can be used for multiplexed assay to study cranial neural crest cell development.

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest ...

Biology

siRNA-Mediated Gene Silencing in Neural Stem Cells

Source: Nguyen, B. H., et al. <a href="https://app.jove.com/v/58346">Culturing and Manipulation of O9-1 Neural Crest Cells.</a> J. Vis. Exp (2018)
This video demonstrates the use of small interfering RNA (siRNA) to silence specific genes of interest in neural stem cells and differentiate them into desired cell types using appropriate differentiation medium. This procedure enables the study of the critical role of specific genes in the differentiation process of neural stem cells.

1. Manipulation of O9-1 cells

	Performing siRNA knockdown in O9-1 cells
	
		Thaw and coat a 24-well plate with a basement membrane matrix before starting ...

Culturing and Manipulation of O9-1 Neural Crest Cells

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