We thank Dr. Ana-Maria Zaske, operator of Atomic Force Microscope-UT Core facility at the University of Texas Health Sciences Center, for the contributed expertise in AFM in this project. We also thank the funding sources from the National Institutes of Health (K01DE026561, R03DE025873, R01DE029014, R56HL142704, and R01HL142704 to J. Wang).

1. Hydrogel preparation
NOTE: All steps must be performed in a cell culture hood that has been disinfected with ethanol and ultraviolet (UV)-sterilized before use to maintain sterility. Tools, such as tweezers and pipettes, must be sprayed with ethanol. Buffer solutions must also be sterile-filtered.
<ol>
	<li>Preparation of aminosilane-coated glass coverslips
	<ol>
		<li>Place the desired number of glass coverslips onto a piece of laboratory wipe. 
		NOTE: Prepare an additional 3-4 coverslips to ensure sufficient backup supplies as they break easily. Different materials of glass coverslips will yield different compatibility of cell seeding and attachment. It is better to determine which type suits the experiment best before starting the experiments (see the Table of Materials).</li>
		<li>Use an alcohol burner or Bunsen burner to sterilize each coverslip by passing it back and forth through the flame (30 s for protein assay experiments). Place each glass coverslip on a laboratory wipe to cool down.</li>
		<li>Once the glass coverslips are cooled down, transfer them onto a Petri dish lined with parafilm to prevent slippage. 
		NOTE: If the coverslips are not cool enough, the residual heat will melt the parafilm onto the slips, rendering them unusable.</li>
		<li>Cover the coverslips with approximately 200 &#956;L and 800 &#956;L of 0.1 M NaOH for a 12 mm and a 25 mm coverslip, respectively, and let them sit for 5 min. Then, aspirate the 0.1 M NaOH and allow the coverslips to air-dry for another 5 min to form an even film.</li>
		<li>Once the coverslips are dried, pipette approximately 80 &#956;L and 150 &#956;L of 3-aminopropyl triethoxysilane (APTS) for 12 mm and 25 mm coverslips, respectively. Be careful to avoid spilling the solution onto the parafilm. Allow the solution to sit for 5 min.</li>
		<li>Aspirate as much excess APTS as possible and allow the residual APTS to dry for 5 min. Rinse the coverslips well by submerging them in sterile, deionized (DI) H2O three times for 5 min each time. 
		NOTE: If the glass coverslips are not rinsed well, the residual APTS causes unwanted reactions with glutaraldehyde, causing a white precipitate to form and resulting in unusable coverslips.</li>
		<li>Move the coverslips to a new Petri dish with the reactive side facing up. Add enough 0.5% glutaraldehyde to the Petri dish to cover the coverslips entirely and allow the coverslips to sit for 30 min.</li>
		<li>Aspirate the 0.5% glutaraldehyde and rinse the coverslips again in DI H2O one time for 3 min. Set the coverslips reactive side up on a laboratory wipe or a clean Petri dish to air-dry completely before using. 
		NOTE: The protocol can be paused here; coverslips must be placed in sterile DI H2O until use.</li>
	</ol>
	</li>
	<li>Preparation of siliconized coverslips
	<ol>
		<li>Place the same number of coverslips as the aminosilane-coated coverslips (step 1.1.1) in a Petri dish lined with parafilm.</li>
		<li>Pipette 40 &#956;L or 150 &#956;L for 12 mm and 25 mm coverslips, respectively, of dichloromethylsilane (DCMS) to one side of the coverslip and allow the solution to sit for 5 min.</li>
		<li>Aspirate any remaining solution from the coverslip, wash in sterile DI H2O once for 1 min, and place the reactive coverslips face up on a laboratory wipe to air-dry completely before moving onto the next step.</li>
	</ol>
	</li>
	<li>Preparing hydrogels
	<ol>
		<li>Mix acrylamide, bis-acrylamide, and DI H2O in a 1.5 mL centrifuge tube to prepare 500 &#956;L of solutions with varying stiffnesses (see Table 1). Vortex the solution for 30 s to mix it thoroughly.</li>
		<li>Working swiftly, add the 10% ammonium persulfate solution (APS) and tetramethylethylenediamine (TEMED) to the tube and vortex the solutions again to mix the solutions. 
		NOTE: Prepare fresh 10% APS and leave it on ice or freeze into single-use aliquots due to its sensitive freeze/thaw cycle.</li>
		<li>Pipette approximately 33 &#956;L or 100 &#956;L of the solution onto the dried 12 mm or 25 mm aminosilane-coated coverslips (section 1.1), respectively.</li>
		<li>Using curved tweezers, immediately place the DCMS-treated coverslip on top of the gel solution with the treated side touching the gel solution, thus sandwiching the gel solution between the DCMS-treated coverslip and aminosilane-coated coverslip.</li>
		<li>Allow the gel solution to polymerize for 5-15 min, while actively monitoring for gel polymerization of the leftover solution in the tube.</li>
		<li>Once the gel is polymerized, separate the DCMS-treated coverslip with curved tweezers or a razor blade, leaving the gel attached to the original aminosilane-coated coverslip.</li>
		<li>Immediately place the coverslip with the attached hydrogel in a predetermined 4-well/24-well and 6-well plate covered with 500 &#181;L and 2 mL of sterile PBS or DI H2O for 12 mm and 25 mm coverslips, respectively, to prevent the gel from drying out.</li>
		<li>Repeat steps 1.3.4-1.3.7 for all coverslips.</li>
		<li>Submerge the hydrogels in sterile PBS or DI H2O for 30 min to remove excess acrylamide solution. Store the hydrogels in sterile PBS or DI H2O at 4 &#176;C for a procedural stop here.</li>
		<li>In a dark room, prepare the sulfosuccinimidyl 6-(4&#39;-azido-2&#39;-nitrophenylamino) hexanoate (sulfo-SANPAH) mixture by mixing 2.5 mL of 50 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid(HEPES) (pH=8.5) with 25 &#956;L of the 50 &#956;g/mL sulfo-SANPAH in a conical tube, wrapped in aluminum foil to protect from light. Use a pipette to mix the solution well before using. 
		NOTE: A volume of 2.5 mL of sulfo-SANPAH solution is enough for approximately twenty-five 12 mm hydrogels or five 25 mm hydrogels.</li>
		<li>Aspirate PBS or DI H2O from the well plate. Add approximately 100 &#956;L or 500 &#956;L for 12 mm and 25 mm coverslips, respectively, of sulfo-SANPAH solution (step 1.3.10) to cover the gel. Ensure the solution covers the gel entirely. 
		NOTE: Adjust the vacuum suction strength to prevent the strong force from ripping or disturbing the hydrogels.</li>
		<li>Place the gels with the solution under a 15 W, 365 nm UV&#160;light for 10 min, uncovered to minimize any interference of the UV light reacting with the sulfo-SANPAH.</li>
		<li>Aspirate the excess sulfo-SANPAH by tilting the plate to collect as much of the solution as possible. Wash the gel with 50 mM HEPES two to three times.</li>
		<li>Add 500 &#956;L and 2 mL for 12 mm and 25 mm gels, respectively, of 50 mg/mL collagen I diluted in 0.2% acetic acid to each well containing the hydrogel. Allow the gels to incubate overnight in a 37 &#176;C, 5% CO2 incubator. 
		NOTE: Dilute collagen I in 0.2% acetic acid instead of 50 mM HEPES to promote homogenous distribution and attachment of collagen I.</li>
		<li>Aspirate the collagen I and wash the gels with sterile PBS three times to remove excess collagen I for 5 min each wash. Incubate the hydrogels in PBS with 10% horse serum, 5% FBS&#160;for 2 h in the 37 &#176;C, 5% CO2 incubator. 
		NOTE: The addition of 10% horse serum promotes higher proliferation compared to only using FBS as done in the previous publication.</li>
		<li>Aspirate the medium. Add 500 mL of sterile-filtered Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% FBS and 1% penicillin-streptomycin (P/S) to each well. Store the gels in the 37 &#176;C, 5% CO2 incubator until ready for cell culture.</li>
		<li>Once ready, plate approximately 1.5 &#215; 104 O9-1 cells/cm2 in basal medium in the culture dishes. Incubate the cells for 2 days in an incubator at 37 &#176;C, 5% CO2. Check the cells for confluency to ensure that the cells are sufficiently attached to gels, and that the number of cells is enough before collecting for analysis. 
		NOTE: See the previously published protocol for the steps of recovery, passage, and collection of O9-1 cells20.</li>
		<li>Proceed to sections 2, 3, or 4 for further analysis of the hydrogels.</li>
	</ol>
	</li>
</ol>
2. Quantitative analysis of stiffness via AFM
<ol>
	<li>Start the AFM system computer, followed by the AFM controller (see the Table of Materials).</li>
	<li>Mount the AFM cantilever on the AFM probe holder. Use a spherical cantilever with a 0.5 &#956;m silica bead mounted at the end of the cantilever (cantilever with spherical bead). 
	NOTE: For stiffer hydrogels, such as 10 kPa, 20 kPa, and 40 kPa, a stiffer probe was used with the spring constant of 0.24 N/m. A softer probe was used for softer hydrogels, such as 0.5 kPa and 1 kPa, with a spring constant of 0.059 N/m.</li>
	<li>Set the AFM software under contact mode.</li>
	<li>Mount the silicon wafer onto the AFM sample stage to collect force curves by clicking on Engage for the cantilever to touch the silicon substrate, thus generating the force curves.</li>
	<li>Use the force curves above (2.4) for calibration, click on Calibrate in the controlling software to obtain an average spring constant of the cantilever under thermal tune condition, and save the calibrated values in the controlling software.</li>
	<li>Mount the samples by placing the coverslip with the attached hydrogel in a 60 mm Petri dish onto the AFM scanning stage. Add 3 mL of PBS into the dish before conducting measurements to prevent the gel from drying out.</li>
	<li>Set the AFM to work in contact mode (fluid) to start measurement. Engage the spherical bead to continuously touch and lift from the gel sample.</li>
	<li>Set the cantilever so that its deflection threshold remains at 10 nm. Keep the ramping size of the probe at 10 &#956;m. Then, record the force curves as in step 2.4.</li>
	<li>Acquire at least 20 force curves from at least 3 to 10 different spots across the surface of the hydrogel.</li>
	<li>Calculate the average Young&#39;s modulus of ~20 force curves for each spot with AFM imaging and analysis software. Use extend ramp force curves and a linearized model (spherical). Calculate the average of all spots for each sample to yield the final stiffness. 
	NOTE: Young&#39;s modulus and related data (i.e., standard deviations) are automatically saved as a spreadsheet.</li>
	<li>Repeat steps 2.6-2.10 for all samples.</li>
</ol>
3. Molecular analysis of stiffness via immunofluorescence staining
<ol>
	<li>Use tweezers to transport the coverslip to a new plate to minimize false signals from cells grown directly onto the plate. Wash the cells with 500 &#181;L of sterile PBS three times to remove dead cells and any remaining culture medium.</li>
	<li>Fix the cells using 500 &#181;L of 4% paraformaldehyde (PFA) for 10 min at room temperature, undisturbed. Then, rewash the cells three times using 500 &#181;L of PBS/well for 2 min each. 
	NOTE: Store at 4 &#176;C for a procedural stop.</li>
	<li>Treat the cells with 500 &#181;L of 0.1% Triton X-100 for 15 min at room temperature. Then, wash the cells three times with 500 &#181;L of PBS/well.</li>
	<li>Block the cells with 250 &#181;L of 10% donkey serum (diluted in PBS and 0.1% Tween 20) per well for 30 min at room temperature.</li>
	<li>Incubate the cells with 250 &#181;L of primary antibodies for 2 h at room temperature or overnight at 4 &#176;C. Then, wash the cells three times with 500 &#181;L of PBS/well for 5 min each. 
	NOTE: Anti-Vinculin (Vcl) (1:250) and anti-AP2 alpha (1:250) were used in this experiment and were diluted in 10% donkey serum.</li>
	<li>Incubate the cells with corresponding secondary antibodies and/or phalloidin used for F-actin staining at a dilution of 1:400 in 250 &#181;L of 10% donkey serum for 30 min at room temperature. Then, wash the cells three times with PBS for 5 min each. 
	NOTE: 568 nm Phalloidin can be co-incubated with 488 nm or 647 nm secondary antibodies or on its own.</li>
	<li>Incubate the cells with 4&#8242;,6-diamidino-2-phenylindole (DAPI, 1:1000 dilution) in 250 &#181;L of PBS for 10 min followed by one last wash of PBS for 2 min.</li>
	<li>Add 3-4 drops of mounting medium to each well. Store the samples at 4 &#176;C to set for at least 2 h before imaging to ensure the mounting medium has set properly.</li>
	<li>Capture images of at least 3 random frames per hydrogel sample with a fluorescence microscope, producing individual and merged channels.</li>
</ol>
4. Quantitative real-time PCR (RT-qPCR)
<ol>
	<li>Transfer the hydrogels with the adherent cells for RNA collection to a new plate to minimize unwanted RNA from cells attached to the cell plate. Wash the cells with PBS three times to remove dead cells and culture medium.</li>
	<li>Extract the total mRNA using an RNA extraction kit. Perform reverse-transcription of RNA to cDNA using a reverse transcription supermix following the manufacturer&#39;s instructions.</li>
	<li>Perform RT-qPCR with primers for Vcl as the stiffness marker of choice and analyze using the 2-&#916;&#916;CT method. 
	NOTE: Primer sequence of Vcl: Forward 5&#39; GCTTCAGTCAGACCCATACTCG 3&#39;; reverse 5&#39; AGGTAAGCAGTAGGTCAGATGT 3&#39;.</li>
</ol>

The authors have no conflicts of interest to disclose.

The goal of the current study is&#160;to provide an effective and efficient in vitro system to better understand the impact of mechanical signals in NCCs. In addition to following the step-by-step protocol mentioned above, researchers need to keep in mind that the cell culture of O9-1 NCCs is affected by the type of glass coverslips used to prepare hydrogels. For instance, it was noted that cells seeded on a specific type of glass coverslip (see the Table of Materials) survived and proliferated ...

Neural crest cells (NCCs) are a group of stem cells during vertebrate embryogenesis with a remarkable ability to migrate and contribute to the development of various organs and tissues. NCCs can differentiate into different cell types, including sensory neurons, cartilage, bone, melanocytes, and smooth muscle cells, depending on the location of axial origin and the local environmental guidance of the NCC1,2. With the ability to differentiate into a wide array of cell types, genetic abnormalities that cause dysregulation at any stage of neural crest (NC) development can lead to numerous congenital diseases2. For instance, perturbations during the formation, migration, and development of NCCs lead to developmental disorders known collectively as neurocristopathies1,3. These diseases range from craniofacial defects due to failure in NCC formation, such as Treacher Collins syndrome, to the development of various cancers due to NCC metastatic migratory ability, as seen in melanoma3,4,5,6. Over the last few decades, researchers have made remarkable discoveries about the roles and mechanisms of NCCs in development and diseases, with the majority of findings being focused on chemical signals7,8. More recently, mechanical signals have been indicated to play a critical but poorly understood role in NCC development9,10.
The environmental cues of NCCs play a critical role during their development, including the regulation of NCC differentiation into various cell types. Environmental cues, e.g., physical cues, influence pivotal behaviors and cellular responses, such as functional diversification. Mechanotransduction allows cells to sense and respond to those cues to maintain various biological processes2. NCCs are surrounded by neighboring cells and different substrates, such as the extracellular matrix (ECM), which can give rise to mechanical stimuli to maintain homeostasis and adapt to the changes through fate determination, proliferation, and apoptosis11. Mechanotransduction begins at the plasma membrane where the sensory component of mechanical extracellular stimuli occurs, resulting in the intracellular regulation of the cell12. Integrins, focal adhesions, and junctions of the plasma membrane relay mechanical signals, such as shearing forces, stress, and the stiffness of surrounding substrates, into chemical signals to produce cellular responses12. The relaying of chemical signals from the plasma membrane to the final cellular regulation is carried out via different signaling pathways to finalize vital processes for the organism, such as differentiation.
Several studies have suggested that mechanical signaling from substrate stiffness plays a role in cell differentiation13,14. For instance, previous studies have shown that mesenchymal stem cells (MSCs) grown on soft substrates with a stiffness similar to that of brain tissue (in the range of 0.1-1.0 kPa) resulted in neuronal cell differentiation15,16. However, more MSCs differentiate into myocyte-like cells when grown on 8-17 kPa substrates mimicking the stiffness of muscle, while osteoblast-like differentiation was observed when MSCs were cultured on stiff substrates (25-40 kPa)15,16. The significance of mechanotransduction is highlighted by the irregularities and abnormalities in the mechanical signaling pathway that potentially lead to severe developmental defects and diseases, including cancer, cardiovascular diseases, and osteoporosis17,18,19. In cancers, normal breast tissue is soft, and the risk of breast cancer increases in stiff and dense breast tissue, an environment that is more akin to breast tumors15. With this knowledge, the effects of mechanical signaling on NCC development can be studied through simple manipulation of substrate stiffness through an in vitro system, providing further advantages and possibilities in understanding the fundamentals of NC-related disease progression and etiology.
To study the impact of mechanical signals in NCCs, we established an efficient in vitro system for NCCs based on the optimization of previously published methods and evaluation of the responses of NCCs to different mechanical signals20,21. A detailed protocol was provided for varying hydrogel stiffness preparation and evaluation of the impact of mechanical signaling in NCCs. To achieve this, O9-1 NCCs are utilized as the NC&#160;model to study the effects and changes in response to stiff versus soft hydrogels. O9-1 NCCs are a stable NC cell line isolated from mouse embryo (E) at day 8.5. O9-1 NCCs mimic NCCs in vivo because they can differentiate into various NC-derived cell types in defined differentiation media22. To study the mechanical signaling of NCCs, a matrix substrate was fabricated with tunable elasticity from varying concentrations of acrylamide and bis-acrylamide solutions to achieve the desired stiffness, correlating to the biological substrate stiffness20,21,23. To optimize the conditions of matrix substrate for NCCs, specifically O9-1 cells, modifications were made from the previously published protocol20. One change made in this protocol was to incubate hydrogels in collagen I, diluted in 0.2% acetic acid instead of 50 mM HEPES, at 37 &#176;C overnight. The low pH of acetic acid leads to a homogeneous distribution and higher collagen I incorporation, thus allowing for a more uniform attachment of the ECM protein24. In addition, a combination of horse serum and fetal bovine serum (FBS) was used at the concentrations of 10% and 5% in phosphate buffer saline (PBS), respectively, before storing the hydrogels in the incubator. Horse serum was used as an additional supplement to FBS due to its ability to promote cell proliferation and differentiation at the concentration of 10%25.
With this method, a biological environment was mimicked by the ECM protein coating (e.g., Collagen I) to create an accurate in vitro environment for NCCs to grow and survive20,21. The stiffness of the prepared hydrogels was quantitatively analyzed via atomic force microscopy (AFM), a well-known technique to depict the elastic modulus26. To study the effect of different stiffness levels on NCCs, wild-type O9-1 cells were cultured and prepared on hydrogels for immunofluorescence (IF) staining against filamentous actin (F-actin) to show the differences in cell adhesion and morphologies in response to changes in substrate stiffness. Utilizing this in vitro system, researchers will be able to study the roles of mechanical signaling in NCCs and its interaction with other chemical signals to gain a deeper understanding of the relationship between NCCs and mechanical signaling.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 mm #1 Corning 0211 Glass Coverslip</td>
			<td>Chemglass Life Sciences</td>
			<td>CLS-1763-012</td>
			<td></td>
		</tr>
		<tr>
			<td>2% Bis-Acrylamide</td>
			<td>Sigma Aldrich</td>
			<td>M1533</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner Bio-one</td>
			<td>662165</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm #1 Corning 0211 Glass Coverslip</td>
			<td>Chemglass Life Sciences</td>
			<td>CLS-1763-025</td>
			<td></td>
		</tr>
		<tr>
			<td>3-aminopropyl triethoxysilane (APTS)</td>
			<td>Sigma Aldrich</td>
			<td>A3648</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well cell culture plate</td>
			<td>Thermo Scientific</td>
			<td>179830</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>J61899-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide</td>
			<td>Sigma Aldrich</td>
			<td>A4058</td>
			<td></td>
		</tr>
		<tr>
			<td>50% glutaraldehyde</td>
			<td>Sigma Aldrich</td>
			<td>G7651</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM cantilever (spherical bead)</td>
			<td>Novascan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AFM software</td>
			<td>Catalyst NanoScope</td>
			<td></td>
			<td>Model: 8.15 SR3R1</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate (APS)</td>
			<td>Sigma Aldrich</td>
			<td>248614</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>anti-AP-2&#945; Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-12726</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Vinculin antibody</td>
			<td>Abcam</td>
			<td>ab129002</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscopy (AFM) Bioscope Catalyst</td>
			<td>Bruker Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type I (100mg)</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermo Fisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethylsilane (DCMS)</td>
			<td>Sigma Aldrich</td>
			<td>440272</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Leica</td>
			<td></td>
			<td>Model DMi8</td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting medium</td>
			<td>SouthernBiotech</td>
			<td>0100-35</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Corning</td>
			<td>35-030-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript Reverse Transcription Supermix</td>
			<td>Bio-Rad</td>
			<td>1708841</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin antibiotic</td>
			<td>Thermo Fisher</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy micro kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 1x PBS</td>
			<td>Hyclone</td>
			<td>SH30256.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile deionized water</td>
			<td>Hardy Diagnostics</td>
			<td>U284</td>
			<td></td>
		</tr>
		<tr>
			<td>sulfo-SANPAH</td>
			<td>Thermo Fisher</td>
			<td>22589</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR green</td>
			<td>Applied Biosystems</td>
			<td>4472908</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized o9-1/hydrogel system for studying mechanical signals in neural crest cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Hydrogel preparation and stiffness assessment through AFM and the Hertz model 
Here, a detailed protocol is provided to generate polyacrylamide hydrogels of varying stiffness by regulating the ratio of acrylamide and bis-acrylamide. However, the polyacrylamide hydrogels are not ready for the adhesion of cells due to the lack of ECM proteins. Thus, sulfo-SANPAH, acting as a linker, covalently binds to the hydrogels and reacts with the primary amines of ECM proteins to allow the adhesion of ECM protei...

Watch this Scientific Journal Video about An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells at JoVE.com

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

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JoVE Journal

Developmental Biology

2.5K Views.  The University of Texas Health Science Center at Houston. Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness. 

Video: An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

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, ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

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 ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...