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 previously established protocol13.
<ol>
	<li>Prepare the basement membrane matrix-coated plate.
	<ol>
		<li>Thaw the basement membrane matrix (see Table of Materials) on ice. Dilute the matrix 1:50 in chilled 1x PBS, and keep on ice.</li>
		<li>Coat a 10 cm culture plate with 10 mL of the diluted basement-membrane matrix solution. Incubate the plate at room temperature for 1 h, and aspirate the matrix before use.</li>
		<li>Wash the plate 3x with 2 mL of PBS.</li>
	</ol>
	</li>
	<li>Culture O9-1 cells on the basement membrane matrix-coated plate.
	<ol>
		<li>Warm the complete embryonic stem (ES) cell medium (see Table of Materials) in a water bath at 37 &#176;C.</li>
		<li>Gently mix the O9-1 cell suspension (see Table of Materials) and count the number of cells using an automated cell counter (see Table of Materials). Adjust the cell concentration to 1.1 &#215; 106/mL with complete ES cell medium.</li>
		<li>Add 8 mL of pre-warmed complete ES cell medium to the matrix-coated 10 cm culture plate, and seed the O9-1 cells at 1.1 x 106 cells/plate. Incubate at 37 &#176;C in a 5% CO2 humidified incubator.</li>
		<li>The next day, replace the medium with fresh complete ES cell medium (pre-warmed to 37 &#176;C). Replace with fresh medium every 2-3 days thereafter. 
		NOTE: When the cells are approximately 80% confluent (3-4 days after plating), they can be dissociated with 0.25% trypsin-EDTA and passaged further or frozen for later use. Representative images of O9-1 cells are shown in Figure 1.</li>
		<li>Wash the culture plate with 2 mL of 1x PBS prior to trypsinization. Add 2 mL of pre-warmed 0.25% trypsin-EDTA and incubate for 5 min at 37 &#176;C. Check for complete cell detachment; gently tap the side of the plate a couple of times if necessary.</li>
		<li>Add 2 mL of pre-warmed complete ES cell medium to the culture plate and transfer the dissociated cells to a 15 mL conical tube. Centrifuge the tube at 300 x g for 5 min.</li>
		<li>Discard the supernatant without disturbing the cell pellet. Then, add 2 mL of pre-warmed complete ES cell medium to the tube, and thoroughly resuspend the cells by pipetting. Seed the cells on a new culture plate at the desired cell density.</li>
		<li>Alternatively, prepare a frozen cell stock by adding 1 mL of cell freezing medium containing 10% dimethyl sulfoxide (DMSO) to the tube in place of complete ES cell medium and thoroughly resuspending the cells. Transfer the cell suspension to cryo tubes, and store at &#8722;80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of the HA/Col1-coated dish
NOTE: The original method of coating HA/Col1 onto glass-bottom dishes was proposed by Irie et al.7.
<ol>
	<li>Carefully add 50 &#181;L of undiluted triethoxysilane to a 3.5 cm glass-bottom dish (see Table of Materials). Incubate for 5 min at room temperature (RT) and protect from light. 
	NOTE: The incubation with triethoxysilane should not exceed 5 min. This may affect the coating efficiency and produce undesirable products.</li>
	<li>Wash the dish quickly 3x with 2 mL of distilled water. Add 50 &#181;L of 0.25% glutaraldehyde, diluted 100x in PBS, per dish, and incubate at RT for 30 min.</li>
	<li>Wash quickly 4x with 2 mL of PBS. Then, coat the dishes with 300 &#181;L of collagen type I in 0.2 N acetic acid at RT for 1 h.</li>
	<li>Wash quickly 3x with 2 mL of PBS. Add 300 &#181;L of 200 &#181;g/mL fluoresceinamine-labeled sodium hyaluronate-H2 (FAHA-H2) (diluted in PBS) to each dish and incubate overnight at RT. Wash again 3x with 2 mL of PBS. After aspirating the PBS, air-dry the plate for 5 min on a clean bench. 
	&#8203;NOTE: FAHA-H2 is a fluoresceinamine-labeled sodium hyaluronate with an average molecular weight ranging from 1,200-1,600 KDa. HA of an appropriate molecular weight can be used according to the research design.</li>
</ol>
3. Migration assay on the HA/Col1-coated dish
NOTE: A wound closure-based assay using defined 500 &#181;m cell-free gaps in Col1/HA substrates was performed using 2-well culture inserts (see Table of Materials). The O9-1 cells express Tmem2, which is required for the adhesion and degradation of HA in the extracellular space9 (Figure 2 and Figure 3).
<ol>
	<li>After drying the coated glass-bottom dish, attach the 2-well culture inserts to the dishes, and fill the inserts externally with 1 mL of PBS.</li>
	<li>Seed the O9-1 cells into the wells at 1 x 104 cells in 100 &#181;L of Dulbecco&#39;s modified Eagle medium (DMEM) containing 2% fetal bovine serum (FBS) per insert. Culture the cells for 2 days at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Remove the inserts carefully from the coated glass-bottom dishes with tweezers. Wash the coated glass-bottom dishes gently with 2 mL of 1x PBS to remove the cells and cell debris. Add 2 mL of fresh DMEM containing 2% FBS into the culture dishes.</li>
	<li>Capture phase-contrast images now as the starting time point using an all-in-one fluorescence microscope in monochrome mode with high-resolution settings (gain at 6 dB, no binning). Objective lenses with magnifications of 4x to 20x were used for imaging in this study (see Table of Materials). 
	NOTE: Capture the phase-contrast images with the corresponding scale bars and save them in TIFF format.</li>
	<li>Culture the cells for an additional 48 h at 37 &#176;C and 5% CO2. Capture phase-contrast images at 24 h and 48 h in culture using the all-in-one fluorescence microscope.</li>
	<li>Fix the cells at 48 h with 1 mL of 4% paraformaldehyde (PFA) for 15 min at room temperature or overnight at 4 &#176;C. Then, wash the dishes 3x for 5 min each with 1 mL of fresh PBS.</li>
	<li>Place a coverslip with mounting medium (see Table of Materials) for further morphological observation. 
	NOTE: The dishes can be imaged immediately or stored for up to 2 months at 4 &#176;C.</li>
	<li>Optional: Immunolabel the cells for proteins of interest. 
	NOTE: A protocol to detect the focal adhesion (FA) complex using a mouse-derived monoclonal anti-vinculin antibody (see Table of Materials) is described here as an example.
	<ol>
		<li>Incubate the dishes (from step 3.6) with 0.5 mL of blocking buffer (5% normal goat serum in PBS) for 60 min. 
		NOTE: Choose an appropriate blocking buffer for the primary antibody. A typical blocking buffer would be 5% normal serum from the same species as the secondary antibody used.</li>
		<li>Prepare the diluted primary antibody in antibody dilution buffer (1% normal goat serum in PBS). Incubate the dishes with 0.5 mL of diluted primary antibody overnight at 4 &#176;C. 
		NOTE: Choose the appropriate antibody dilution buffer. Typically, the antibody is used at a 1:50-1:200 dilution.</li>
		<li>Rinse 3x for 5 min each with 2 mL of PBS. Prepare the diluted goat-derived anti-mouse secondary antibody in the antibody dilution buffer (1% normal goat serum in PBS). Incubate the dishes with 0.5 mL of diluted secondary antibody for 1-3 h at room temperature. 
		NOTE: Typically, the secondary antibody is used at a 1:500-1:1,000 dilution. DAPI can be used for staining the nuclei.</li>
		<li>Rinse 3x with 2 mL of PBS for 5 min each time. Place the coverslip with 50 &#181;L of mounting medium. 
		NOTE: The dishes can be imaged using the all-in-one fluorescence microscope with a GFP filter (excitation: 470/40, emission: 525/50) and a TexasRed filter (excitation: 560/40, emission: 630/75) in monochrome mode with high-resolution settings (gain at 6 dB, no binning). Objective lenses with magnifications of 4x to 20x were used for the imaging in this study. 
		&#8203;NOTE: The slide can be stored for up to 2 months at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Open the ImageJ 1.51s software. In the ImageJ window, select File &#62; Open from the menu bar to open the saved image file.</li>
	<li>For setting the measurement scale, draw a line of the same distance as the scale bar. Go to Analyze &#62; Set scale and type the known distance and units of the line in the appropriate boxes of the Set scale window.</li>
	<li>Draw a straight line between the cell gap, and press Analyze &#62; Measure to transfer the values to a data window. Measure at least five different positions in each sample, and average the distances to obtain the representative sample data. Perform the statistical analyses using appropriate software (see Table of Materials).</li>
</ol>

<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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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).
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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 ...

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

Research

JoVE Journal

Developmental Biology

949 Views.  Osaka University Graduate School of Dentistry. 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.

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

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

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

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

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

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

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

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Chapters in this video

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using Chick^GFPNeural Tube Grafting and Carbocyanine Dye DiI Injection

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

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