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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Craniofacial+birth+defects:+The+role+of+neural+crest+cells+in+the+etiology+and+pathogenesis+of+Treacher+Collins+syndrome+and+the+potential+for+prevention.">Craniofacial birth defects: The role of neural crest cells in the etiology and pathogenesis of Treacher Collins syndrome and the potential for prevention.</a> American Journal of Medical Genetics. Part A. 152 (12), 2984-2994 (2010).</li><li>Bronner, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+pattern+of+cell+fate+restrictions+in+the+neural+crest+lineage.">Timing and pattern of cell fate restrictions in the neural crest lineage.</a> Development. 124 (21), 4351-4359 (1997).</li><li>Harris, M. L., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+specification+in+neural+crest+cell+pathfinding.">Lineage specification in neural crest cell pathfinding.</a> Developmental Dynamics. 236 (1), 1-19 (2007).</li><li>Luo, R., Gao, J., Wehrle-Haller, B., Henion, P. 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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. F. <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:+An+emerging+regulator+of+craniofacial+and+dental+development.">Hippo pathway: An emerging regulator of craniofacial and dental development.</a> Journal of Dental Research. 96 (11), 1229-1237 (2017).</li><li>Pan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hippo+signaling+pathway+in+development+and+cancer.">The hippo signaling pathway in development and cancer.</a> Developmental Cell. 19 (4), 491-505 (2010).</li><li>Xiao, Y., Leach, J., Wang, J., Martin, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway+in+organ+development,+homeostasis,+and+regeneration.">The Hippo pathway in organ development, homeostasis, and regeneration.</a> Current Opinion in Cell Biology. 49, 99-107 (2017).</li><li>Moya, I. M., Halder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway+in+cellular+reprogramming+and+regeneration+of+different+organs.">The Hippo pathway in cellular reprogramming and regeneration of different organs.</a> Current Opinion in Cell Biology. 43, 62-68 (2016).</li><li>Piccolo, S., Dupont, S., Cordenonsi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+YAP/TAZ:+hippo+signaling+and+beyond.">The biology of YAP/TAZ: hippo signaling and beyond.</a> Physiological Reviews. 94 (4), 1287-1312 (2014).</li><li>Bauer, D. E., Canver, M. C., Orkin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+genomic+deletions+in+mammalian+cell+lines+via+CRISPR/Cas9.">Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9.</a> Journal of Visual Experiments : JoVE. (95), e52118 (2015).</li><li>Ran, F. A., 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=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Manderfield, L. 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=Hippo+signaling+is+required+for+Notch-dependent+smooth+muscle+differentiation+of+neural+crest.">Hippo signaling is required for Notch-dependent smooth muscle differentiation of neural crest.</a> Development. 142 (17), 2962-2971 (2015).</li><li>Hindley, C. 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=The+Hippo+pathway+member+YAP+enhances+human+neural+crest+cell+fate+and+migration.">The Hippo pathway member YAP enhances human neural crest cell fate and migration.</a> Scientific Reports. 6, 23208 (2016).</li><li>Nejigane, S., Haramoto, Y., Okuno, M., Takahashi, S., Asashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+coactivators+Yap+and+TAZ+are+expressed+during+early+Xenopus+development.">The transcriptional coactivators Yap and TAZ are expressed during early Xenopus development.</a> International Journal of Developmental Biology. 55 (1), 121-126 (2011).</li><li>Grefte, S., Vullinghs, S., Kuijpers-Jagtman, A. M., Torensma, R., Von den Hoff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel,+but+not+collagen+I,+maintains+the+differentiation+capacity+of+muscle+derived+cells+in+vitro.">Matrigel, but not collagen I, maintains the differentiation capacity of muscle derived cells in vitro.</a> Biomedical Materials. 7 (5), 055004 (2012).</li><li>Kang, B. 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=Effect+of+matrigel+on+the+osteogenic+potential+of+canine+adipose+tissue-derived+mesenchymal+stem+cells.">Effect of matrigel on the osteogenic potential of canine adipose tissue-derived mesenchymal stem cells.</a> Journal of Veterinary Medical Science. 74 (7), 827-836 (2012).</li><li>Uemura, 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=Matrigel+supports+survival+and+neuronal+differentiation+of+grafted+embryonic+stem+cell-derived+neural+precursor+cells.">Matrigel supports survival and neuronal differentiation of grafted embryonic stem cell-derived neural precursor cells.</a> Journal of Neuroscience Research. 88 (3), 542-551 (2010).</li></ol>

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 200mM (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&#39;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) 100X</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100mM)</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) 106 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&#38;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&#39;s salts, ribonucleosides, deoxyribonucleosides, &#38; 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...

Watch this Scientific Journal Video about Culturing and Manipulation of O9-1 Neural Crest Cells at JoVE.com

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

9.7K Views.  Baylor College of Medicine. 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.

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

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...