In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration
Summary
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.
Abstract
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.
Introduction
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 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.
Protocol
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.
- Prepare the basement membrane matrix-coated plate.
- Thaw the basement membrane matrix (see Table of Materials) on ice. Dilute the matrix 1:50 in chilled 1x PBS, and keep on ice.
- 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.
- Wash the plate 3x with 2 mL of PBS.
- Culture O9-1 cells on the basement membrane matrix-coated plate.
- Warm the complete embryonic stem (ES) cell medium (see Table of Materials) in a water bath at 37 °C.
- 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 × 106/mL with complete ES cell medium.
- 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 °C in a 5% CO2 humidified incubator.
- The next day, replace the medium with fresh complete ES cell medium (pre-warmed to 37 °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. - 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 °C. Check for complete cell detachment; gently tap the side of the plate a couple of times if necessary.
- 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.
- 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.
- 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 −80 °C.
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.
- Carefully add 50 µL 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. - Wash the dish quickly 3x with 2 mL of distilled water. Add 50 µL of 0.25% glutaraldehyde, diluted 100x in PBS, per dish, and incubate at RT for 30 min.
- Wash quickly 4x with 2 mL of PBS. Then, coat the dishes with 300 µL of collagen type I in 0.2 N acetic acid at RT for 1 h.
- Wash quickly 3x with 2 mL of PBS. Add 300 µL of 200 µg/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.
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.
3. Migration assay on the HA/Col1-coated dish
NOTE: A wound closure-based assay using defined 500 µm 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).
- 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.
- Seed the O9-1 cells into the wells at 1 x 104 cells in 100 µL of Dulbecco's modified Eagle medium (DMEM) containing 2% fetal bovine serum (FBS) per insert. Culture the cells for 2 days at 37 °C in a 5% CO2 humidified incubator.
- 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.
- 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. - Culture the cells for an additional 48 h at 37 °C and 5% CO2. Capture phase-contrast images at 24 h and 48 h in culture using the all-in-one fluorescence microscope.
- Fix the cells at 48 h with 1 mL of 4% paraformaldehyde (PFA) for 15 min at room temperature or overnight at 4 °C. Then, wash the dishes 3x for 5 min each with 1 mL of fresh PBS.
- 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 °C. - 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.- 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. - 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 °C.
NOTE: Choose the appropriate antibody dilution buffer. Typically, the antibody is used at a 1:50-1:200 dilution. - 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. - Rinse 3x with 2 mL of PBS for 5 min each time. Place the coverslip with 50 µL 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.
NOTE: The slide can be stored for up to 2 months at 4 °C.
- Incubate the dishes (from step 3.6) with 0.5 mL of blocking buffer (5% normal goat serum in PBS) for 60 min.
4. Data analysis
- Open the ImageJ 1.51s software. In the ImageJ window, select File > Open from the menu bar to open the saved image file.
- For setting the measurement scale, draw a line of the same distance as the scale bar. Go to Analyze > Set scale and type the known distance and units of the line in the appropriate boxes of the Set scale window.
- Draw a straight line between the cell gap, and press Analyze > 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).
Representative Results
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).
Figure 1: TMEM2 expression in NCCs. Transverse sections of the neural tube of Tmem2-FLAGKI embryos at E9.0. (A) Sections at the cranial and trunk levels of the neural tube were double-labeled for the TMEM2-FLAG protein and HA. TMEM2 expression was observed in the neural plate and the border region of the neural tube (filled arrowheads), whereas these sites were devoid of HA staining (open arrowheads). (B) Double-labeling of the neural crest cells for TMEM2-FLAG and Sox9. Transverse sections of the E9.0 neural tube were stained for TMEM2-FLAG and Sox9. Sox9-positive pre-migratory and emigrating NCCs at the edge of the neural tube highly expressed TMEM2. Abbreviation: nt = neural tube. Scale bars: (A) 300 µm; (B) 100 µm. Adapted with permission from Inubushi et al.9. Please click here to view a larger version of this figure.
Figure 2: Degradation of HA by TMEM2 in O9-1 cells. (A) Representative images of Tmem2-depleted and control O9-1 cells cultured on a regular culture dish (left). (B) Expression of Tmem2 in these cells was evaluated by qPCR, with Gapdh as an internal control for normalization (bar graph). Means ± SD (n = 5) are shown as horizontal bars. ***p < 0.001 by unpaired Student's t-test. Scale bar, 5.0 µm. (C) Cell-based hyaluronidase assay. Tmem2-depleted and control O9-1 cells were cultured for 48 h on glass coverslips coated with fluoresceinated HA (FA-HA). HA degrading activity is revealed as dark areas in the fluorescent background. The level of HA degradation was also quantitatively compared between Tmem2-depleted and control O9-1 cells as described in Materials and Methods (bargraph). Data represent mean ± SD of the fluorescence intensity underneath a cell relative to that in the cell-free area (n > 50 cells per condition pooled from three independent experiments). ***p < 0.001 by unpaired Student's t-test. Adopted with permission from Inubushi et al.9. Please click here to view a larger version of this figure.
Figure 3: Degradation of substrate-bound HA at FAs in O9-1 cells. Cell-based hyaluronidase assays were performed for 16 h and cells were stained for vinculin. In control O9-1 cells, HA degradation occurs in vinculin-positive FAs. In Tmem2-depleted O9-1 cells, HA degradation and FA formation are greatly diminished. The number of FAs per cell was quantitatively compared between Tmem2-depleted and control O9-1 cells (bar graph). Data represent mean ± SD (n >30 cells per condition pooled from three independent experiments). ***p < 0.001 by unpaired Student's t-test. Adopted with permission from Inubushi et al.9. Please click here to view a larger version of this figure.
Figure 4: Representative images of O9-1 cell migration into a cell-free gap on mixed Col1/HA substrates. (A) The top panel shows the gaps at the start of the experiment (Day 0). The bottom panels show gap images after 24 h (Day 1) or 48 h (Day 2) incubation. Scale bar = 150 µm. (B) A bar graph showing the quantitative analysis of cell migration. The data represent the mean ± SD of the gap area covered by migratory cells relative to the area of the original gap (n = 5 per condition). *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired Student's t-test. Please click here to view a larger version of this figure.
Figure 5: Degradation of HA in the mixed Col1/HA substrates at FAs in O9-1 cells. O9-1 cells cultured on mixed substrates consisting of Col1/HA were immunolabeled with anti-vinculin antibody (red). The dark spots/streaks represent HA degradation activity in the FAHA-H2 substrate (green). The sites of HA degradation and focal adhesions (vinculin) were co-localized on the mixed substrates (arrowheads). Scale bar = 500 µm. Please click here to view a larger version of this figure.
Discussion
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 migration during neural development17. Other ECM components, including laminin and fibronectin, influence NCC adhesion and migration18. However, the specific roles of ECM components in NCC migration are still unknown.
The in vitro model described here aims to examine the behavior of migrating NCCs encountering HA-rich ECM. We chose 2D, rather than 3D, substrates due to the difficulties in preparing composite collagen-HA gels with uniform gelation properties. In a 2D culture system, the spatial gradients of the soluble factor concentrations and the stiffnesses of the extracellular matrix substrates are different from those of live tissue19. From this point of view, a 3D culture system has great advantages and can provide deeper insights into cell migration mechanisms20,21. In this context, setting up a 3D assay system is preferred, if applicable. This in vitro experimental model may be applicable to examining NCC migration behavior in response to varying ECM components. This would enable a better understanding of the mechanistic basis of NCC migration, which governs embryo morphogenesis.
The O9-1 cells used in this study stably express stem cell markers (CD44, Sca-1, and Bmi1) and neural crest markers (AP-2a, Twist1, Sox9, Myc, Ets1, Dlx1, Dlx2, Crabp1, Epha2, and Itgb1). Under non-differentiating culture conditions, O9-1 cells maintain multipotent stem cell-like characteristics. O9-1 cells have the potential to differentiate into multiple cell types, including osteoblasts, chondrocytes, smooth muscle cells, and glial cells under specific culture conditions12. Therefore, O9-1 cells are a useful tool for investigating the molecular properties of cranial NCC migration and differentiation. Nevertheless, the use of primary NCCs (e.g., from chicken or mouse embryonic neural tubes) instead of O9-1 cells will provide more detailed data to facilitate a better understanding of the nature of NCC migration.
In the wound closure-based migration assay, the NCC will also proliferate and the increase in the cell number will also contribute to the expansion of the cell population into the gap area22. To minimize the proliferation during wound healing assay, serum starvation is the most common method instead of using pharmacological agents23. However, the effects of serum starvation should be tested in each cell type.
The most critical step of this protocol is adding the covalent coating of HA to the glass-bottom dishes. Glass surface treatment with silane reagents is used for the analysis of focal adhesions to ECM substrates24. Triethoxysilane is an organosilicon compound containing many free amino groups that can bind to free hydroxyl groups on silica glass surfaces25. A technique was developed and optimized in this study to produce a thin, stable silane layer on a silica glass surface using triethoxysilane. The amine in the triethoxysilane-coated glass surface can bind the carboxyl groups on HA, consequently chemically immobilizing HA on the glass surface8. However, with this method, it is not possible to bind other protein-based ECM substrates to the glass surface. Therefore, glutaraldehyde was used here to chemically immobilize the type I collagen through amine coupling to the aldehyde. Notably, inadequate HA coating can lead to the removal of the HA coating even without enzymatic digestion (e.g., by the action of mechanical force during cell adhesion and migration). Coating with ECM substrates in place of collagen type I is possible, provided the substrates possess amine groups. However, limitations may exist in covalently coating the ECM onto glass-bottom dishes due to the chemical properties of the substrate; thus, trial and error may be required.
Although not a perfect model to mimic NCC migration into the HA-rich ECM surrounding the neural tube, this in vitro migration experiment may be useful for the functional analysis of the molecules involved in in vivo NCC migration. In addition, this in vitro assay may be useful for therapeutic drug screening to accelerate or prevent HA degradation, as well as the migration of other cell types, including skin fibroblasts and keratinocytes.
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.). 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.
Materials
| 10cm cell culture dish | CORNING | Cat. 353003 | |
| 1X PBS | Millipore | Cat. No. BSS-1005-B | |
| 2-well culture inserts | ibidi | Cat. No. 80209 | |
| Alexa 555-labelled goat anti-mouse IgG | Invitrogen | Cat. A21422 | Goat derived anti-mouse secondary antibody |
| automated cell counter | Bio-Rad | Cat. No. TC20 | |
| CELLBANKER | ZENOGEN PHARMA | Cat. 11910 | Cell freezing medium |
| collagen type I | Sigma | Cat. No. 08-115 | |
| Complete ES Cell Medium | Millipore | Cat. No. ES-101-B | |
| DAPI | Invitrogen | Cat. 10184322 | |
| Dulbecco’s Modified Eagle Medium | Gibco | Cat. 11971025 | |
| Fetal Bovine serum | Gibco | Cat. 10270106 | |
| fluorescence microscope | Keyence | Cat. No. BZ-X700 | |
| Fluoresent labelled HA | PG Research | FAHA-H2 | |
| Glas bottom dish | Iwaki | Cat. 11-0602 | |
| glutaldehyde | Sigma | Cat. No. G5882 | |
| Matrigel | Fisher | Cat. No. CB-40234 | The basement-membrane matrix |
| monoclonal anti-vinculin antibody | Sigma | Cat. No. V9264 | |
| mounting media | Dako | S3023 | |
| Normal goat serum | Fisher | Cat. 50062Z | |
| O9-1 cells | Millipore | Cat. No. SCC049 | |
| Paraformaldehyde | Sigma | Cat. 158127 | |
| triethoxysilane | Sigma | Cat. No. 390143 | |
| trypsin-EDTA | Millipore | Cat. No. SM-2003-C |
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