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) lethality^4,5,6.

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 sites^7,8. 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.

1. Prepare the basement membrane matrix-coated plate.
   1. Thaw the basement membrane matrix (see Table of Materials) on ice. Dilute the matrix 1:50 in chilled 1x PBS, and keep on ice.
   2. 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.
   3. Wash the plate 3x with 2 mL of PBS.
2. Culture O9-1 cells on the basement membrane matrix-coated plate.
   1. Warm the complete embryonic stem (ES) cell medium (see Table of Materials) in a water bath at 37 °C.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. 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.
   8. 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.

1. 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.
2. 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.
3. 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.
4. 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).

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
   1. 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.
   2. 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.
   3. 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.
   4. 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.

4. Data analysis

1. Open the ImageJ 1.51s software. In the ImageJ window, select File > Open from the menu bar to open the saved image file.
2. 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.
3. 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).

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).

Discussion

Various ECM components regulate NCC emigration/migration. For instance, HA positively regulates NCC migration^2,15. 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...

Materials

Name	Company	Catalog Number	Comments
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