I express great acknowledgment to Fumitoshi Irie and Yu Yamaguchi for their encouragement and kind suggestions in establishing this method. This work was supported by grants-in-aid for scientific research programs from the Japan Society for the Promotion of Science (#19KK0232 to T.I., #20H03896 to T.I.).&#160;The original method for the coating of HA onto glass substrates and in situ HA degradation assays on the substrates was described in Yamamoto et al. (2017)8, while the method for the preparation of HA/Col1 mixed substrates was described in Irie et al. (2021)7.

All procedures were approved by the Animal Ethics Committee of the Osaka University Graduate School of Dentistry.
1. Culture of mouse cranial neural crest cells
NOTE: The neural crest cell line used in this study comprises O9-1 cells, originally derived from Wnt1-Cre; R26R-GFP-expressing cells isolated from E8.5 mouse embryos12 (see discussion). The method described here for culturing O9-1 cells follows a prev...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versican+is+selectively+expressed+in+embryonic+tissues+that+act+as+barriers+to+neural+crest+cell+migration+and+axon+outgrowth.">Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth.</a> Development. 121 (8), 2303-2312 (1995).</li><li>Camenisch, T. D., 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=Disruption+of+hyaluronan+synthase-2+abrogates+normal+cardiac+morphogenesis+and+hyaluronan-mediated+transformation+of+epithelium+to+mesenchyme.">Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme.</a> Journal of Clinical Investigation. 106 (3), 349-360 (2000).</li><li>Camenisch, T. D., Schroeder, J. A., Bradley, J., Klewer, S. E., McDonald, J. 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E., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+manipulation+of+O9-1+neural+crest+cells.">Culturing and manipulation of O9-1 neural crest cells.</a> Journal of Visualized Experiments. (140), e58346 (2018).</li><li>Humphries, J. D., 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=Vinculin+controls+focal+adhesion+formation+by+direct+interactions+with+talin+and+actin.">Vinculin controls focal adhesion formation by direct interactions with talin and actin.</a> Journal of Cell Biology. 179 (5), 1043-1057 (2007).</li><li>Perris, R., Perissinotto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+extracellular+matrix+during+neural+crest+cell+migration.">Role of the extracellular matrix during neural crest cell migration.</a> Mechanisms of Development. 95 (1-22), 3-21 (2000).</li><li>Perris, R., 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=Spatial+and+temporal+changes+in+the+distribution+of+proteoglycans+during+avian+neural+crest+development.">Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development.</a> Development. 111 (2), 583-599 (1991).</li><li>Zagris, N., Gilipathi, K., Soulintzi, N., Konstantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decorin+developmental+expression+and+function+in+the+early+avian+embryo.">Decorin developmental expression and function in the early avian embryo.</a> The International Journal of Developmental Biology. 55 (6), 633-639 (2011).</li><li>Perris, R., Paulsson, M., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+avian+neural+crest+cell+migration+on+fibronectin+and+laminin.">Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin.</a> Developmental Biology. 136 (1), 222-238 (1989).</li><li>Chevalier, N. 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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=Capturing+complex+3D+tissue+physiology+in+vitro.">Capturing complex 3D tissue physiology in vitro.</a> Nature Reviews Molecular Cell Biology. 7 (3), 211-224 (2006).</li><li>Jonkman, J. E., 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=An+introduction+to+the+wound+healing+assay+using+live-cell+microscopy.">An introduction to the wound healing assay using live-cell microscopy.</a> Cell Adhesion and Migration. 8 (5), 440-451 (2014).</li><li>Davis, P. K., Ho, A., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+methods+for+cell-cycle+synchronization+of+mammalian+cells.">Biological methods for cell-cycle synchronization of mammalian cells.</a> Biotechniques. 30 (6), 1322-1331 (2001).</li><li>Wayner, E. A., Garcia-Pardo, A., Humphries, M. J., McDonald, J. A., Carter, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+the+T+lymphocyte+adhesion+receptor+for+an+alternative+cell+attachment+domain+(CS-1)+in+plasma+fibronectin.">Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin.</a> Journal of Cell Biology. 109 (3), 1321-1330 (1989).</li><li>Robert, G. 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The author declares that no competing interests exist.

Various ECM components regulate NCC emigration/migration. For instance, HA positively regulates NCC migration2,15. Interestingly, a study based on genetic mouse models of Tmem2, a cell surface hyaluronidase, elucidated the requirement of HA degradation in NCC migration9. Collagens are also abundant in the ECM surrounding the neural tube16. Decorin, a small leucine-rich proteoglycan, has been shown to regulate NCC mi...

Neural crest cells (NCCs) are a multipotent cell population that is present in developing embryos, and they originate from the neural plate border during neurulation. They contribute to the formation of a variety of tissues, including the peripheral nervous system, cardiovascular system, craniofacial tissues, and the skeleton1. After induction and NCC specification at the neural plate border, NCCs emigrate from the neuroepithelium and migrate toward NCC-derived tissue sites1.
Hyaluronan (HA) is a non-sulfated glycosaminoglycan that is distributed in a variety of tissues as a component of the extracellular matrix (ECM). The importance of HA in embryo development has been demonstrated in model systems through the ablation of genes responsible for hyaluronan metabolism. For instance, mutations in hyaluronan synthase genes (Has1 and Has2) in Xenopus were found to lead to NCC migration defects and craniofacial malformation2. In addition, the HA-binding proteoglycans, aggrecan and versican, have been reported to exert inhibitory effects on NCC migration3. In mice, Has2 ablation leads to severe defects in endocardial cushion formation, resulting in mid-gestation (E9.5-10) lethality4,5,6.
Transmembrane protein 2 (Tmem2), a cell surface hyaluronidase, has been recently demonstrated to play a critical role in promoting integrin-mediated cancer cell adhesion and migration by removing matrix-associated HA at the adhesion sites7,8. More recently, Inubushi et al.9 demonstrated that a deficiency in&#160;Tmem2 leads to severe craniofacial defects due to abnormalities in NCC emigration/migration and survival. In the previous study9, Tmem2 expression was analyzed during NCC formation and migration. Tmem2 expression was observed at the site of NCC delamination and in emigrating Sox9-positive NCCs (Figure 1). Additionally, using Tmem2-depleted mouse O9-1 neural crest cells, the study demonstrated that the in vitro expression of Tmem2 was essential for the O9-1 cells to form focal adhesions and for their migration into HA-containing substrates (Figure 2 and Figure 3)9.
These results strongly indicate that Tmem2 is also important for NCC adhesion and migration through the HA-rich ECM. However, the molecular mechanism of NCC adhesion and migration within the HA-rich ECM is still unclear. It is, therefore, necessary to establish an in vitro culture experimental system to fully explore NCC adhesion and migration within the HA-rich ECM.
Of the numerous approaches employed in testing cell migration, the cell wound closure-based assay is a simple method frequently used in the fields of physiology and oncology10. This approach is useful due to its relevance to the in vivo phenotype and is effective in determining the roles of drugs and chemoattractants during cell migration11. It is possible to evaluate the migration ability of both whole cell masses and individual cells by measuring the cell gap distances over time11. In this manuscript, a modified in vitro wound closure-based assay is introduced to model NCC migration into HA-rich tissues surrounding the neural tube. This procedure is also applicable for studying different ECM components (i.e., collagens, fibronectin, and laminin) to analyze the role of the ECM scaffold in NCC migration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10cm cell culture dish</td>
			<td>CORNING</td>
			<td>Cat. 353003</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Millipore</td>
			<td>Cat. No. BSS-1005-B</td>
			<td></td>
		</tr>
		<tr>
			<td>2-well culture inserts</td>
			<td>ibidi</td>
			<td>Cat. No. 80209</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 555-labelled goat anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>Cat. A21422</td>
			<td>Goat derived anti-mouse secondary antibody</td>
		</tr>
		<tr>
			<td>automated cell counter</td>
			<td>Bio-Rad</td>
			<td>Cat. No. TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLBANKER</td>
			<td>ZENOGEN PHARMA</td>
			<td>Cat. 11910</td>
			<td>Cell freezing medium</td>
		</tr>
		<tr>
			<td>collagen type I</td>
			<td>Sigma</td>
			<td>Cat. No. 08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete ES Cell Medium</td>
			<td>Millipore</td>
			<td>Cat. No. ES-101-B</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>Cat. 10184322</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium&#160;</td>
			<td>Gibco</td>
			<td>Cat. 11971025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>Gibco</td>
			<td>Cat. 10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Keyence</td>
			<td>Cat. No. BZ-X700</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresent labelled HA</td>
			<td>PG Research</td>
			<td>FAHA-H2</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom dish</td>
			<td>Iwaki</td>
			<td>Cat. 11-0602</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaldehyde</td>
			<td>Sigma</td>
			<td>Cat. No. G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Fisher</td>
			<td>Cat. No. CB-40234</td>
			<td>The basement-membrane matrix</td>
		</tr>
		<tr>
			<td>monoclonal anti-vinculin antibody</td>
			<td>Sigma</td>
			<td>Cat. No. V9264</td>
			<td></td>
		</tr>
		<tr>
			<td>mounting media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Fisher</td>
			<td>Cat. 50062Z</td>
			<td></td>
		</tr>
		<tr>
			<td>O9-1 cells</td>
			<td>Millipore</td>
			<td>Cat. No. SCC049</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>Cat. 158127</td>
			<td></td>
		</tr>
		<tr>
			<td>triethoxysilane</td>
			<td>Sigma</td>
			<td>Cat. No. 390143</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Millipore</td>
			<td>Cat. No. SM-2003-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A migration assay was performed on mixed substrates composed of Col1 and high-molecular weight HA (average molecular weight: 1,200-1,400 kDa) using the protocol described here. O9-1 cells at the boundary of the gap were found to readily migrate into the HA-rich gap (Figure 4). Immunostaining for a FA marker, vinculin14, confirmed that the O9-1 cells formed focal adhesions (FAs) at the sites of HA degradation (Figure 5).
<p class="jove...

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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.

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 ...

Hyaluronic acid is one of the most abundant extracellular matrix in animal model. The importance of hyaluronic acid in embryonic development has been demonstrated. This in vitro model is useful to explore how neural crest cells adhere and migrate within the hyaluronic acid-rich extracellular matrix.With our technique, we can evaluate the migration and the HA degradation ability of both wholesale population and then individual cells over time. In addition, this technique can be used for drug screening to find compound that accelerate or prevent HA degradation. To begin, dilute the basement membrane matrix with chilled PBS at a 1:50 ratio and keep it on ice.Coat a 10 centimeter culture plate with 10 milliliters of the diluted matrix solution and incubate the plate at room temperature for one hour. Wash the plate three times with two milliliters of PBS. To culture the O9-1 cells on the matrix coated platem warm the complete embryonic stem or ES cell medium in a water bath at 37 degrees Celsius.Gently mix the O9-1 cell suspension and count the number of cells using an automated cell counter. Then adjust the cell concentration to 1.1 million cells per milliliter with the complete ES cell medium. Add eight milliliters of prewarmed complete ES cell medium to the matrix coated 10 centimeter culture plate and seed the O9-1 cells at 1.1 million cells per plate.Incubate at 37 degrees Celsius in a 5%carbon dioxide humidified incubator. The next day, replace the medium with fresh complete ES cell medium prewarmed to 37 degrees Celsius. Replace with fresh medium every two to three days thereafter.Wash the culture plate with two milliliters of PBS and then add two milliliters of prewarmed 0.25%Trypsin-EDTA. Incubate for five minutes at 37 degrees Celsius. Add two milliliters of prewarmed complete ES cell medium to the culture plate and transfer the dissociated cells to a 15 milliliter conical tube.Centrifuge the tube at 300g for five minutes and discard the supernatant without disturbing the cell pellet. Add two milliliters of prewarmed complete ES cell medium to the tube and thoroughly resuspend the cells by pipetting. Then seed the cells on a new culture plate at the desired cell density.Carefully add 50 microliters of undiluted triethoxysilane to a 3.5 centimeter glass bottom dish and incubate for five minutes at room temperature protected from light. Wash the dish three times with two milliliters of distilled water and add 50 microliters of 0.25%glutaraldehyde diluted 100 times in PBS per dish. Incubate at room temperature for 30 minutes then wash four times with two milliliters of PBS and coat the dishes with 300 microliters of collagen type one in 0.2 normal acetic acid at room temperature for one hour.Again, wash three times with two milliliters of PBS. Add 300 microliters of 200 micrograms per milliliter fluoresceinamine, labeled sodium hyaluronate H2 to each dish and incubate overnight at room temperature. Wash again with two milliliters of PBS three times.After drying the coated glass bottom dish, attach the two well culture inserts to the dishes and fill the inserts externally with one milliliter of PBS. Seed the O9-1 cells into the wells at 10, 000 cells in 100 microliters of DMEM containing 2%fetal bovine serum or FBS per insert. Culture the cells for two days at 37 degrees Celsius in a 5%carbon dioxide humidified incubator, Capture phase contrast images as the starting time point, using an all-in-one fluorescence microscope.Remove the inserts carefully from the coated glass bottom dishes with tweezers and gently wash the coated glass bottom dishes with two milliliters of PBS to remove the cells and cell debris. Add two milliliters of fresh DMEM containing 2%FBS into the cultured dishes. Culture the cells for an additional 48 hours at 37 degrees Celsius and 5%carbon dioxide and capture phase contrast images after 24 hours and 48 hours in culture.Wash the dishes with PBS and fix the cells after 48 hours with one milliliter of 4%paraformaldehyde for 15 minutes at room temperature or overnight at four degrees Celsius. Then wash the dishes three times for five minutes each with one milliliter of fresh PBS. Finally, place a cover slip with the mounting medium for further morphological observation.Transverse sections of the neural tube of Tmem2 flag embryos at E9.0 are shown in this figure. The sections at the cranial and trunk levels of the neural tube were double labeled for the Tmem2 flag protein and hyaluronan. Tmem2 expression was observed in the neural plate and the border region of the neural tube whereas these sites were devoid of hyaluronan staining.Double labeling of the neural crest cells for Tmem2 flag and Sox9 is shown here. Transverse sections of the E9.0 neural tube were stained for Tmem2 flag and Sox9. The representative images of Tmem2 depleted and control O9-1 cells cultured on a regular culture dish are shown in this figure.The expression of Tmem2 in these cells was evaluated by qPCR with GAPDH as an internal control for normalization. Tmem2 depleted and control O9-1 cells were cultured for 48 hours on glass cover slips coated with fluoresceinated hyaluronan. Hyaluronan degrading activity is revealed as dark areas in the fluorescent background.The level of hyaluronan degradation was also quantitatively compared between Tmem2 depleted and control O9-1 cells. The degradation of substrate-bound hyaluronan at the focal adhesions in O9-1 cells is shown in this figure. O9-1 cells cultured on mixed substrates consisting of Col1/HA were immuno labeled with an anti-vinculin antibody.The dark spots or streaks represent hyaluronan degradation activity in the FAHA H2 substrate. The sites of hyaluronan degradation and focal adhesions were co-localized on the mixed substrates. Coval coating the glass bottom dishes with HA is a critical step in the protocol because inadequate coating can result in the HA easily removed by mechanical force during adhesion and migration.To ensure proper coating, we use glutaraldehyde, chemically-immobilized type one collagen to the glass via arming coupling to the aldehyde. It is possible to use extracellular matrix substrate beside type one collagen, but they must have arming groups and there may be limitations in coval coating them on to glass bottom dishes due to their chemical properties. So trial and error may be necessary to determine the best approach.This lab experiment can be helpful in studying how certain molecule work during the movement of neural crest cells into the HA or HA environment around the neural tube. It could also be a useful way to test drug that could either accelerate or prevent this migration process. It is interesting to know why Tmem2 has both the functions of adhesion over HA and the degradation of HA and why HA need to be degradated at the full cohesion site.Since HA is the most common substance in the extracellular matrix, figuring out the answer to this question is really important in biology and medicine. This experimental protocol is valuable because it can be applied to various cell types, including skin fibroblasts, epithelial cells, and cancer cells.

in-vitro-investigation-effects-hyaluronan-rich-extracellular-matrix

Research

JoVE Journal

Developmental Biology

917 Views.  Osaka University Graduate School of Dentistry. Hyaluronic acid is one of the most abundant extracellular matrix in animal model. The importance of hyaluronic acid in embryonic development has been demonstrated. This in vitro model is useful to explore how neural crest cells adhere and migrate within the hyaluronic acid-rich extracellular matrix.With our technique, we can evaluate the migration and the HA degradation ability of both wholesale population and then individual cells over time. In addition, this technique can be used for...