Name: Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay (Video) | JoVE Journal
Uploaded: 2025-02-14T14:40:00.000+00:00
Duration: 8 min 55 s
Description: Learn a three-dimensional in vitro protocol for differentiating cranial neural crest cells from mouse embryonic stem cells. This method reduces neurosphere size variability and enables multiplexed assays for developmental biology research.

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5 minutes

We thank Dr. Remi Xavier Coux for advice on primer design and expertise in cell culture. This work was supported by the European Research Council (ERC Starting Grant 101039995 - REGENECREST) and the Fondation pour la Recherche M&#233;dicale (Amor&#231;age - AJE202205015403).

1. Generation of a single-cell suspension from mouse ESC colonies
NOTE: This protocol is adapted to the use of CK35 mESC (an mESC line competent for germ line transmission, to have then the option to develop in vivo models15) grown on inactivated feeders in a gelatin-coated TC-treated 6-well plate. One well of a TC-treated 6-well plate should yield approximately 1.5 &#215; 106 mESC, which is sufficient for the rest of the protocol. This can be scaled up if necessary. Adjust the initial steps in accordance with the chosen ESC strain and maintenance culture method, as well as the proper culture medium. This protocol is to be performed under sterile conditions. See the Table of Materials for details related to all the materials, reagents, and equipment used in this protocol.
<ol>
	<li>Start from mESC at a 70%-80% confluence grown in mESC culture medium. See Table 1 for the mESC culture medium composition used in this study. 
	NOTE: Do not let mESC grow over 80% confluence, as they will start to differentiate, and this will affect the aggregation process. Colonies must be compact and show a healthy morphology (no cracks or waves, distinct nuclear-cytoplasm contrast).</li>
	<li>Prepare CNCC differentiation medium. See Table 1 for CNCC differentiation medium composition. 
	NOTE: Once growth factors are added, the medium can be stored for up to 3 weeks at 4 &#176;C. Ensure the medium is protected from light.</li>
	<li>Prepare a fresh collagenase solution at a concentration of 2 mg/mL in the DMEM-Knockout medium. 
	NOTE: Use of collagenase ensures only the colonies are detached and not the feeders, as their presence in the following steps will interfere with NS aggregation. Filter collagenase solution before use with a 0.22 &#181;m filter.</li>
	<li>Aspirate the ESC medium from mESC.</li>
	<li>Gently add 1 mL of PBS to the side of the well. Rock the plate gently to ensure even washing.</li>
	<li>Remove PBS and replace with 2 mL of collagenase solution. Incubate at 37 &#176;C for 30-45 min.
	<ol>
		<li>Check the plate under a light microscope at 10x magnification after the first 20 min and then every 5 min.</li>
		<li>When the colonies show rolled-up edges, tap vigorously on the plate side, and the colonies will detach.</li>
	</ol>
	</li>
	<li>With a 5 mL serological pipette, collect the colonies and transfer them to a 15 mL conical tube. 
	NOTE: Check the plate under a light microscope for leftover colonies. These can be collected with a PBS wash.</li>
	<li>Centrifugate the colonies at 16 &#215; g for 3 min at room temperature (RT).</li>
	<li>Aspirate as much medium as possible from the conical tube, being careful not to disturb the colonies at the bottom of the tube.</li>
	<li>Add 1 mL of 0.05% trypsin solution and incubate the tube at 37 &#176;C for 5 min.</li>
	<li>Dissociate colonies in the tube by pipetting vigorously up and down, first with a p1000 and then with a p200 micropipette. 
	NOTE: This ensures the obtention of a single-cell suspension.</li>
	<li>Add 2 mL of mESC culture medium to block the trypsin, and centrifugate at 160 &#215; g for 3 min at RT. Remove the supernatant and add 1 mL of CNCC differentiation medium.</li>
	<li>Count cells using an automated cell counting device&#160;or a standardized system under the microscope, following manufacturer instructions.</li>
	<li>After counting, dilute with sufficient CNCC differentiation medium to obtain a concentration of 3000 live cells per 50 &#181;L.</li>
	<li>Using a p200 micropipette, seed 50 &#181;L of the cell suspension in each well of a non-TC treated U-bottom 96-well plate, then top up the well until 200 &#181;L with CNCC differentiation medium. 
	NOTE: While filling the plate, resuspend from time to time the single cell suspension with a p1000 micropipette to obtain a homogeneous concentration.</li>
	<li>Incubate overnight in an incubator at 37 &#176;C, 5% CO2.</li>
</ol>
2. Transferring into a flat-bottom 96-well plate for CNCC differentiation
<ol>
	<li>The next day (day 1), observe the plate under a light microscope. Ensure one small cell cluster with clear borders is visible at the bottom of each well. Put the plate back in the incubator overnight. 
	NOTE: There may be some cells around the main aggregate, some dead. This will not interfere with NS aggregation.</li>
	<li>On day 2, slowly remove 100 &#181;L of medium from each well.
	<ol>
		<li>When aspirating medium, be careful not to remove the NS. To avoid doing so, place the pipette tip close to the surface and far from the bottom.</li>
	</ol>
	</li>
	<li>Cut the tip of a p200 micropipette at around 3-4 mm from the tip. Use this to aspirate the NS with the remaining medium. 
	NOTE: To facilitate picking up the NS, gently pipette up and down a couple of times before aspirating.</li>
	<li>Transfer NS and the remaining medium into a non-TC treated flat-bottom 96-well plate and verify the transfer under a light microscope, then top each new well with 100 &#181;L of prewarmed CNCC differentiation medium. Leave NS in the incubator at 37 &#176;C, 5% CO2, until day 4.</li>
	<li>On day 4, remove 100 &#181;L of medium from each well and replace it with 100 &#181;L of prewarmed CNCC differentiation medium. 
	NOTE: To avoid disturbing NS attachment at the bottom of the plate, slowly aspirate and replace medium.</li>
	<li>On days 5 and 6, check the NS attachment under the light microscope. Ensure lighter cells - delaminating from the NS -start surrounding the main body of the NS.</li>
	<li>On day 7, change the medium as described in 2.5. Change the medium with the same procedure every 2 days until the endpoint of the study.</li>
</ol>
3. CNCC passaging and maintenance
NOTE: CNCC passaging can be performed as soon as there is a sufficient quantity of cells visible around NS. This can be as early as day 7, as earlier time points do not provide a sufficient amount of CNCC.
<ol>
	<li>Prepare CNCC Maintenance medium. See Table 1 for CNCC Maintenance medium composition.
	<ol>
		<li>Before adding it to the medium, filter BSA after solubilization through a 0.22 &#956;m filter. Once growth factors are added, store the medium for up to 3 weeks at 4 &#176;C. Ensure the medium is protected from light.</li>
	</ol>
	</li>
	<li>Prepare fibronectin at 7.5 &#181;g/mL in PBS. Mix vigorously.</li>
	<li>Coat the wells of a non-TC treated 96-well plate by adding 100 &#956;L of fibronectin solution per well. Let coat under the hood for 30 min at RT. 
	NOTE: If CNCC are meant for immunofluorescence staining, place a sterile glass coverslip at the bottom of the well before coating. Ensure that the slide stays at the bottom of the well and does not float to avoid coating the opposite side to the one that will be used. Follow the instructions in section 5 for CNCC fixation and mounting.</li>
	<li>In the meantime, in the non-TC treated flat-bottom 96-well plate, aspirate as much CNCC differentiation medium as possible from the wells and substitute with 50 &#181;L accutase. Incubate at 37 &#176;C for 5 min.</li>
	<li>After incubation, add 100 &#181;L of CNCC maintenance medium per well to quench the accutase. Remove fibronectin from the wells of the receiving non-TC treated flat-bottom 96-well plate. Filter the detached post-migratory CNCC by passing them through a 40 &#181;m filter on top of the well of the receiving non-TC treated flat-bottom 96-well plate. 
	NOTE: This will filter out cell clumps or remaining NS that have not been previously removed. CNCC grown in the same condition can be pooled in one 50 mL tube to be then seeded in the same well of a TC-treated 6-well plate. In this case, coat the well with 1 mL of fibronectin and use 1 mL of CNCC maintenance medium&#160;to quench the accutase.</li>
	<li>Let post-migratory CNCC attach for 15-30 min at 37 &#176;C. Discard medium&#160;and replace with 100 &#181;L of CNCC maintenance medium. Change medium&#160;every 2 days. 
	NOTE: Gently add medium as fast flow induces differentiation of CNCC into neural derivatives. If working in a 6-well format, use 1 mL of CNCC maintenance medium.</li>
</ol>
4. NS fixation and mounting for immunofluorescence
<ol>
	<li>At the desired time point, transfer NS from the non-TC treated flat-bottom 96-well plate into a DNA low binding 2 mL tube by cutting the tip of a p200 micropipette and gently pipet up and down to pick up the NS. 
	NOTE: At later time points (day 7 onwards), NS will be big enough to be seen by the eye but also will be harder to detach.</li>
	<li>Let NS settle in the tube for 3 min at RT, remove as much medium as possible, and rinse with 1 mL of cold PBS. 
	NOTE: If transferring small NS from early time points (before days 3-4), spin at 16 &#215; g for 3 min to ensure NS settle to the bottom.</li>
	<li>Remove PBS and, in a chemical hood, replace with 2 mL of 4% PFA in PBS. Incubate for 20 min at RT.
	<ol>
		<li>Invert the tube slowly after adding 4% PFA solution and let it rest during fixation on the side. The goal is to spread NS slightly so they do not stick together in this step.</li>
	</ol>
	</li>
	<li>Remove 4% PFA solution (in the chemical hood) and wash NS with 1 mL of cold PBS/0.5% Tween20. Let it settle at RT for 3 min. Repeat this 3 times in total. 
	NOTE: NS will settle at the bottom.</li>
	<li>Remove PBS/0.5% Tween20 and add 2 mL of PBS/0.1% Triton X-100. Invert the tube and let it rest on the side. Incubate for 1 h at RT.</li>
	<li>Wash NS 3 times in cold PBS/0.5% Tween20, as indicated in step 4.4.</li>
	<li>Remove PBS/0.5% Tween20 and block with 2% BSA in PBS at 4 &#176;C for a minimum of 1 h, preferably overnight. 
	NOTE: Samples can be stored in 2% BSA/PBS for up to 1 week at 4 &#176;C. Protect from light.</li>
	<li>Prepare primary antibody solution by adding the correct dilution of primary antibody to 2% BSA/PBS in a final volume of 500 &#181;L. For the primary antibody mix used in this study, see Table 2.</li>
	<li>Remove as much of the blocking solution as possible and transfer the NS to a 0.5 mL tube by cutting the tip of a p200 micropipette.</li>
	<li>Add the primary antibody solution and pipette up and down slowly to resuspend the NS. Incubate overnight on a rotator at 4 &#176;C.</li>
	<li>Wash NS 3 times in cold PBS for 5 min at RT.</li>
	<li>Prepare the secondary antibody solution by diluting the secondary antibodies of choice in 2% BSA/PBS and adding 1/1000 DAPI. For the secondary antibody mix used in this study, see Table 2. 
	NOTE: Keep the tubes protected from light.</li>
	<li>Replace PBS with 500 &#181;L of secondary antibody solution and wrap the tube with aluminum foil to protect it from light. Incubate for 1 h on the rotator at RT.</li>
	<li>Wash NS 3 times in cold PBS, as indicated in step 4.11.</li>
	<li>Remove as much PBS as possible without aspirating the NS, substitute with 50 &#181;L of clearing agent, and follow manufacturer instructions.</li>
	<li>Incubate overnight at RT, protected from light.</li>
	<li>Prepare mounting chambers.
	<ol>
		<li>On a microscope slide, place three layers of double-sided tape. Use transparent, fibreless tape to avoid residues in the mounting chamber.</li>
		<li>Using a razor, cut a 3 mm &#215; 8 mm window in the double tape. 
		NOTE: Chamber dimensions depend on the sample time point. This example is suited for hosting 50 &#181;L of mounting medium, which is optimal for 10-20 single NS at later time points (days 7-9).</li>
	</ol>
	</li>
	<li>Under a stereoscope, carefully transfer the NS in the clearing agent into the mounting chamber by cutting a low adhesion p200 micropipette tip. Use a magnification between 2x and 4x to provide a field of view of the whole chamber and enough magnification to identify the NS. If the stereoscope is outfitted for fluorescent imaging, working with a blue fluorescence filter will make identifying the NS easier, as the DAPI staining will make the otherwise transparent NS stand out. 
	NOTE: Verify that the medium forms a slight convex meniscus above the chamber. This will ensure there are no bubbles in the chamber.</li>
	<li>Place a coverslip on the surface and lightly press on its sides to make it adhere. Perform this step under the stereoscope and verify that the NS are not pushed outside the chamber.</li>
	<li>Store at 4 &#176;C protected from light until imaging.</li>
</ol>
5. CNCC fixation and mounting for immunofluorescence
<ol>
	<li>Remove CNCC maintenance medium&#160;from the wells and wash them with PBS. Add PBS gently by pipetting on the side of the well.</li>
	<li>Proceed with fixation, permeabilization, and staining as described in steps 4.3-4.14.</li>
	<li>Mount coverslips on a microscopy glass slide by adding mounting medium onto the slide, grabbing and rotating the coverslip with the use of tweezers so that post-migratory CNCC face the glass slide, and placing it on the drop.
	<ol>
		<li>Optional: Seal the coverslip with nail polish or other commonly employed sealing systems.</li>
	</ol>
	</li>
	<li>Store at 4 &#176;C protected from light until imaging.</li>
</ol>

<ol><li>Rothstein, M., Bhattacharya, D., Simoes-Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+basis+of+neural+crest+axial+identity%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">The molecular basis of neural crest axial identity.</a> Dev Biol. 444, S170-S180 (2018).</li>
<li>Smeriglio, P., Zalc, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cranial+neural+crest+cells+contribution+to+craniofacial+bone+development+and+regeneration%5BTitle%5D)+AND+%22Curr+Osteoporos+Rep%22%5BJournal%5D)">Cranial neural crest cells contribution to craniofacial bone development and regeneration.</a> Curr Osteoporos Rep. 21 (5), 624-630 (2023).</li>
<li>Zalc, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactivation+of+the+pluripotency+program+precedes+formation+of+the+cranial+neural+crest%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Reactivation of the pluripotency program precedes formation of the cranial neural crest.</a> Science. 371 (6529), eabb4776(2021).</li>
<li>Perera, S. N., Kerosuo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+road+again%3A+Establishment+and+maintenance+of+stemness+in+the+neural+crest+from+embryo+to+adulthood%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">On the road again: Establishment and maintenance of stemness in the neural crest from embryo to adulthood.</a> Stem Cells. 39 (1), 7-25 (2021).</li>
<li>Nguyen, B. H., Ishii, M., Maxson, R. E., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culturing+and+manipulation+of+O9-1+neural+crest+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Culturing and manipulation of O9-1 neural crest cells.</a> J Vis Exp. (140), e58346(2018).</li>
<li>Ishii, M., Arias, A. C., Liu, L., Chen, Y. B., Bronner, M. E., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+stable+cranial+neural+crest+cell+line+from+mouse%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">A stable cranial neural crest cell line from mouse.</a> Stem Cells Dev. 21 (17), 3069-3080 (2012).</li>
<li>Rada-Iglesias, A., Bajpai, R., Prescott, S., Brugmann, S. A., Swigut, T., Wysocka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenomic+annotation+of+enhancers+predicts+transcriptional+regulators+of+human+neural+crest%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Epigenomic annotation of enhancers predicts transcriptional regulators of human neural crest.</a> Cell Stem Cell. 11 (5), 633-648 (2012).</li>
<li>Cederquist, G. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specification+of+positional+identity+in+forebrain+organoids%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Specification of positional identity in forebrain organoids.</a> Nat Biotechnol. 37 (4), 436-444 (2011).</li>
<li>Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S. A., Flynn, R. A., Wysocka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+unique+chromatin+signature+uncovers+early+developmental+enhancers+in+humans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A unique chromatin signature uncovers early developmental enhancers in humans.</a> Nature. 470 (7333), 279-283 (2011).</li>
<li>Prescott, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancer+divergence+and+cis-regulatory+evolution+in+the+human+and+chimp+neural+crest%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Enhancer divergence and cis-regulatory evolution in the human and chimp neural crest.</a> Cell. 163 (1), 68-83 (2015).</li>
<li>Chaddah, R., Arntfield, M., Runciman, S., Clarke, L., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+neural+stem+cells+from+human+embryonic+stem+cell+colonies%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Clonal neural stem cells from human embryonic stem cell colonies.</a> J Neurosci. 32 (23), 7771-7781 (2012).</li>
<li>Bajpai, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CHD7+cooperates+with+PBAF+to+control+multipotent+neural+crest+formation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">CHD7 cooperates with PBAF to control multipotent neural crest formation.</a> Nature. 463 (7283), 958-962 (2010).</li>
<li>Sipahi, R., Zupanc, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stochastic+cellular+automata+model+of+neurosphere+growth%3A+Roles+of+proliferative+potential%2C+contact+inhibition%2C+cell+death%2C+and+phagocytosis%5BTitle%5D)+AND+%22J+Theor+Biol%22%5BJournal%5D)">Stochastic cellular automata model of neurosphere growth: Roles of proliferative potential, contact inhibition, cell death, and phagocytosis.</a> J Theor Biol. 445, 151-165 (2018).</li>
<li>Mori, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+neurosphere+size+on+the+growth+rate+of+human+neural+stem%2Fprogenitor+cells%5BTitle%5D)+AND+%22J+Neurosci+Res%22%5BJournal%5D)">Effect of neurosphere size on the growth rate of human neural stem/progenitor cells.</a> J Neurosci Res. 84 (8), 1682-1691 (2006).</li>
<li>Kress, C., Vandormael-Pournin, S., Baldacci, P., Cohen-Tannoudji, M., Babinet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonpermissiveness+for+mouse+embryonic+stem+%28ES%29+cell+derivation+circumvented+by+a+single+backcross+to+129%2FSv+strain%3A+establishment+of+ES+cell+lines+bearing+the+Omd+conditional+lethal+mutation%5BTitle%5D)+AND+%22Mamm+Genome%22%5BJournal%5D)">Nonpermissiveness for mouse embryonic stem (ES) cell derivation circumvented by a single backcross to 129/Sv strain: establishment of ES cell lines bearing the Omd conditional lethal mutation.</a> Mamm Genome. 9 (12), 998-1001 (1998).</li>
<li>Simões-Costa, M., Bronner, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishing+neural+crest+identity%3A+a+gene+regulatory+recipe%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Establishing neural crest identity: a gene regulatory recipe.</a> Development. 142 (2), 242-257 (2015).</li>
<li>Memberg, S. P., Hall, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dividing+neuron+precursors+express+neuron-specific+tubulin%5BTitle%5D)+AND+%22J+Neurobiol%22%5BJournal%5D)">Dividing neuron precursors express neuron-specific tubulin.</a> J Neurobiol. 27 (1), 26-43 (1995).</li>
<li>Kim, C. N., Shin, D., Wang, A., Nowakowski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+molecular+dynamics+of+the+developing+human+thalamus%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Spatiotemporal molecular dynamics of the developing human thalamus.</a> Science. 382, eadf9941(2023).</li>
<li>Grimaldi, A., Comai, G., Mella, S., Tajbakhsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+bipotent+progenitors+that+give+rise+to+myogenic+and+connective+tissues+in+mouse%5BTitle%5D)+AND+%22ELife%22%5BJournal%5D)">Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse.</a> ELife. 11, e70235(2022).</li>
<li>To, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multiomic+atlas+of+human+early+skeletal+development%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A multiomic atlas of human early skeletal development.</a> Nature. 635 (8039), 657-667 (2024).</li>
<li>Bajpai, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+stages+of+rapid+and+uniform+neuralization+of+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">Molecular stages of rapid and uniform neuralization of human embryonic stem cells.</a> Cell Death Differ. 16 (6), 807-825 (2009).</li>
<li>Kerosuo, L., Nie, S., Bajpai, R., Bronner, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crestospheres%2C+Long-term+maintenance+of+multipotent%2C+premigratory+neural+crest+stem+cells%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Crestospheres, Long-term maintenance of multipotent, premigratory neural crest stem cells.</a> Stem Cell Reports. 5 (4), 499-507 (2015).</li>
<li>Abe, S., Yamaguchi, S., Sato, Y., Harada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sphere-derived+multipotent+progenitor+cells+obtained+from+human+oral+mucosa+are+enriched+in+neural+crest+cells%5BTitle%5D)+AND+%22Stem+Cells+Transl+Med%22%5BJournal%5D)">Sphere-derived multipotent progenitor cells obtained from human oral mucosa are enriched in neural crest cells.</a> Stem Cells Transl Med. 5 (1), 117-128 (2016).</li>
<li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strengths+and+limitations+of+the+neurosphere+culture+system%5BTitle%5D)+AND+%22Mol+Neurobiol%22%5BJournal%5D)">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li>
<li>Ziegler, L., Grigoryan, S., Yang, I. H., Thakor, N. V., Goldstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+generation+of+Schwann+cells+from+human+embryonic+stem+cell-derived+neurospheres%5BTitle%5D)+AND+%22Stem+Cell+Rev+Rep%22%5BJournal%5D)">Efficient generation of Schwann cells from human embryonic stem cell-derived neurospheres.</a> Stem Cell Rev Rep. 7 (2), 394-403 (2011).</li>
</ol>

The authors declare no conflict of interest.

In vitro 3D differentiation models allow analyzing complex cell interactions that could be difficult - or could not - be observed in 2D cell culture. Several models have been developed to study CNCC development in vitro. These are generally directly derived from ESC colonies7,21 or tissue explants22,23. Although these systems have proven efficient for generating neural crest cells, such ...

Cranial neural crest cells (CNCC) are a stem-like cell population that arises in the anteriormost part of the developing embryo, at the border between the neural plate and the surface ectoderm1. CNCC then undergo an epithelial-to-mesenchymal transition (EMT), delaminate from the neuroepithelium, and migrate dorsoventrally towards various locations in the embryo where they differentiate into a wide variety of cell types2. Studying this cell population is of great interest as it possesses a remarkable plasticity3 and the unique ability to differentiate into both ectodermal and mesenchymal derivatives, such as craniofacial bones and cartilages4. Although CNCC are relatively accessible in the embryo, they are a transient population with a low number of cells, making systemic mechanistic studies difficult to conduct in vivo. CNCC cell lines have been isolated and characterized in the last few years to overcome these limitations. In particular, the O9-1 CNCC cell line is a great tool for studying migratory and post-migratory neural crest development5,6; however, this cell line does not allow the study of the early events prior to migration leading to neural crest induction and specification. In this regard, there have been significant developments in the development of in vitro differentiation protocols to differentiate CNCC in a dish via the use of 3D structures resembling the developing neuroepithelium called neurospheres (NS)7,8- obtained after differentiation of embryonic stem cell (ESC) colonies. These 3D protocols robustly produce high numbers of CNCC, allowing the conduct of biochemical and genomic mechanistic studies9,10. NS are cultured on low attachment plates in N2B27 supplemented medium, together with Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF)10,11 to stimulate cell proliferation. These protocols are carried out in Petri dishes, cultivating numerous NS in the same plate. Within the growing NS, cells aggregate and continue to divide - reaching a diameter of 100-200 &#181;m upon maturity. At maturity (about day 5), NS attach to the substrate and differentiate into CNCC resembling their in vivo counterparts9,12. These CNCC then undergo EMT and delaminate onto the plate surface. Morphological differences can be observed depending on NS size, as larger spheres will appear darker in the core due to lower availability of nutrients and oxygen, leading to cells undergoing apoptosis13. While this type of procedure generates a large number of CNCC at the endpoint of differentiation, it presents several limitations, making the study of the various molecular dynamics occurring during the differentiation process nearly impossible. First, the use of ESC colonies - which vary in size - makes it difficult to control the starting cell number for each experiment. This results in the generation of NS of various shapes and diameters that develop differently by activating specific signaling pathways, leading to altered cell differentiation and, thus not forming a uniform sample at a given time point. Second, culturing multiple NS in the same plate often leads to them fusing together14 and potentially releasing signaling molecules that influence their neighbors' microenvironment and, thus, their development. Altogether, these procedures generate a lot of variability between samples and experiments.
Here, we present a strategy to overcome these difficulties that generate single NS - capable of producing CNCC - by aggregating mouse ESC (mESC) in non-TC treated U-bottomed 96-well plates. Starting from mESC allows studying the specification process and early stages of CNCC development compared to starting from already established neural crest cell lines. This protocol begins with the disaggregation of mESC colonies to obtain a single cell suspension, followed by the seeding of a specific number of mESC in each well of a non-TC treated U-bottomed 96-well plate. The cells are left to aggregate for two days and subsequently moved to a non-TC treated flat-bottomed 96-well plate, in which NS will be able to attach to the plate bottom. By controlling the starting cell number and the microenvironment of each NS during the differentiation process, this protocol reduces sample variability, which increases experimental reproducibility. We believe this will be a convenient platform for designing multiplexed experiments, such as testing the effect of different culture conditions or performing gene perturbation screens.

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cranial neural crest cells three-dimensional in vitro differentiation protocol for multiplexed assay

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.

Following the protocol, mESC colonies were dissociated, and 3000 cells were seeded in non-TC treated U-bottom 96-well plates. On day 2, aggregated NS were transferred into non-TC treated flat-bottom 96-well plates to allow them to attach. A simplified visualization of the NS aggregation protocol is provided in Figure 1A. NS were cultured until day 9 and then processed for immunofluorescence staining. Cells that migrated from the NS onto the plate were transferred to coverslips for imaging an...

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 ...

cranial-neural-crest-cells-three-dimensional-vitro-differentiation

Research

JoVE Journal

Biology

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.

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

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.

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

This video illustrates a technique for isolating cranial neural folds from the midbrain region of chick embryos. It presents a detailed dissection process of the neural folds, their placement, and attachment on fibronectin-coated coverslips, followed by the observation of neural crest cell migration from the neural folds.

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

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.

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

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

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.

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

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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.

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Learn a three-dimensional in vitro protocol for differentiating cranial neural crest cells from mouse embryonic stem cells. This method reduces neurosphere size variability and enables multiplexed assays for developmental biology research.

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

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