Name: modeling paracrine noncanonical wnt signaling in vitro
Uploaded: 2021-12-10T14:30:00
Description: Watch this Scientific Journal Video about Modeling Paracrine Noncanonical Wnt Signaling In Vitro at JoVE.com

This work was supported in part by NIH awards F30HL154324 to O.T. and K08HL121191 and R03HL154301 to S.R.K. The authors would like to acknowledge that the schematic in Figure 1 in this manuscript was created with biorender.com.

1. Preexperimental expansion and passaging of cells
<ol>
	<li>C2C12 cell culture:
	<ol>
		<li>Prepare 500 mL of C2C12 culture medium by combining Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% fetal bovine serum (FBS) and 1 % penicillin/streptomycin.</li>
		<li>Thaw a vial of C2C12 cells in 37 &#176;C water bath. While the C2C12 cells are thawing, add 5 mL of C2C12 medium to a 15 mL conical tube. Immediately transfer the thawed cells to the 15 mL tube using a P1000 pipette. 
		NOTE: C2...

<ol>
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(8), 42093 (2019).</li><li>Li, 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=Planar+cell+polarity+signaling+regulates+polarized+second+heart+field+morphogenesis+to+promote+both+arterial+and+venous+pole+septation.">Planar cell polarity signaling regulates polarized second heart field morphogenesis to promote both arterial and venous pole septation.</a> Development. 146 (20), 181719 (2019).</li><li>Lutze, G., 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=Noncanonical+WNT-signaling+controls+differentiation+of+lymphatics+and+extension+lymphangiogenesis+via+RAC+and+JNK+signaling.">Noncanonical WNT-signaling controls differentiation of lymphatics and extension lymphangiogenesis via RAC and JNK signaling.</a> Scientific Reports. 9 (1), 4739 (2019).</li><li>Buttler, K., 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=Maldevelopment+of+dermal+lymphatics+in+Wnt5a-knockout-mice.">Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice.</a> Developmental Biology. 381 (2), 365-376 (2013).</li><li>Betterman, K. L., 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=Atypical+cadherin+FAT4+orchestrates+lymphatic+endothelial+cell+polarity+in+response+to+flow.">Atypical cadherin FAT4 orchestrates lymphatic endothelial cell polarity in response to flow.</a> Journal of Clinical Investigation. 130 (6), 3315-3328 (2020).</li><li>Descamps, B., 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=Frizzled+4+regulates+arterial+network+organization+through+noncanonical+Wnt/planar+cell+polarity+signaling.">Frizzled 4 regulates arterial network organization through noncanonical Wnt/planar cell polarity signaling.</a> Circulation Research. 110 (1), 47-58 (2012).</li><li>Weeraratna, A. T., 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=Wnt5a+signaling+directly+affects+cell+motility+and+invasion+of+metastatic+melanoma.">Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma.</a> Cancer Cell. 1 (3), 279-288 (2002).</li><li>Henry, C., 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=Expression+of+the+novel+Wnt+receptor+ROR2+is+increased+in+breast+cancer+and+may+regulate+both+&#946;-catenin+dependent+and+independent+Wnt+signalling.">Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both &#946;-catenin dependent and independent Wnt signalling.</a> Journal of Cancer Research and Clinical Oncology. 141 (2), 243-254 (2014).</li><li>Anastas, J. N., 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=A+protein+complex+of+SCRIB,+NOS1AP+and+VANGL1+regulates+cell+polarity+and+migration,+and+is+associated+with+breast+cancer+progression.">A protein complex of SCRIB, NOS1AP and VANGL1 regulates cell polarity and migration, and is associated with breast cancer progression.</a> Oncogene. 31 (32), 3696-3708 (2012).</li><li>Niehrs, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+world+of+WNT+receptor+signalling.">The complex world of WNT receptor signalling.</a> Nature Reviews Molecular Cell Biology. 13 (12), 767-779 (2012).</li><li>Dong, B., 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=Functional+redundancy+of+frizzled+3+and+frizzled+6+in+planar+cell+polarity+control+of+mouse+hair+follicles.">Functional redundancy of frizzled 3 and frizzled 6 in planar cell polarity control of mouse hair follicles.</a> Development. 145 (19), (2018).</li><li>Bernascone, I., 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=Sfrp3+modulates+stromal-epithelial+crosstalk+during+mammary+gland+development+by+regulating+Wnt+levels.">Sfrp3 modulates stromal-epithelial crosstalk during mammary gland development by regulating Wnt levels.</a> Nature Communications. 10 (1), 2481 (2019).</li><li>Hendrickx, G., 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=WNT16+requires+G&#945;+subunits+as+intracellular+partners+for+both+its+canonical+and+noncanonical+WNT+signalling+activity+in+osteoblasts.">WNT16 requires G&#945; subunits as intracellular partners for both its canonical and noncanonical WNT signalling activity in osteoblasts.</a> Calcified Tissue International. 106 (3), 294-302 (2020).</li><li>Avgustinova, A., 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=Tumour+cell-derived+Wnt7a+recruits+and+activates+fibroblasts+to+promote+tumour+aggressiveness.">Tumour cell-derived Wnt7a recruits and activates fibroblasts to promote tumour aggressiveness.</a> Nature Communications. (7), 10305 (2016).</li><li>Tseng, J. C., 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=CAPE+suppresses+migration+and+invasion+of+prostate+cancer+cells+via+activation+of+noncanonical+Wnt+signaling.">CAPE suppresses migration and invasion of prostate cancer cells via activation of noncanonical Wnt signaling.</a> Oncotarget. 7 (25), 38010-38024 (2016).</li><li>Wang, Q., 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=A+novel+role+for+Wnt/Ca2++signaling+in+actin+cytoskeleton+remodeling+and+cell+motility+in+prostate+cancer.">A novel role for Wnt/Ca2+ signaling in actin cytoskeleton remodeling and cell motility in prostate cancer.</a> PLoS One. 5 (5), 10456 (2010).</li><li>Gibbs, B. C., 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=Prickle1+mutation+causes+planar+cell+polarity+and+directional+cell+migration+defects+associated+with+cardiac+outflow+tract+anomalies+and+other+structural+birth+defects.">Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects.</a> Biology Open. 5 (3), 323-335 (2016).</li><li>Cui, C., 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=a+PCP+protein+required+for+ciliogenesis,+regulates+directional+cell+migration+and+cell+polarity+by+direct+modulation+of+the+actin+cytoskeleton.">a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.</a> PLoS Biology. 11 (11), 1001720 (2013).</li><li>Gombos, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formin+DAAM+functions+as+molecular+effector+of+the+planar+cell+polarity+pathway+during+axonal+development+in+Drosophila.">The formin DAAM functions as molecular effector of the planar cell polarity pathway during axonal development in Drosophila.</a> The Journal of Neuroscience. 35 (28), 10154-10167 (2015).</li><li>Toubat, O., 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=Neural+Crest+Cell-derived+Wnt5a+Regulates+Planar+Cell+Polarity+in+Cranial+Second+Heart+Field+Progenitor+Cells.">Neural Crest Cell-derived Wnt5a Regulates Planar Cell Polarity in Cranial Second Heart Field Progenitor Cells.</a> Circulation. 142, 12540 (2020).</li><li>Li, 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=Spatial+regulation+of+cell+cohesion+by+Wnt5a+during+second+heart+field+progenitor+deployment.">Spatial regulation of cell cohesion by Wnt5a during second heart field progenitor deployment.</a> Developmental Biology. 412 (1), 18-31 (2016).</li><li>Sinha, T., 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=Loss+of+Wnt5a+disrupts+second+heart+field+cell+deployment+and+may+contribute+to+OFT+malformations+in+DiGeorge+syndrome.">Loss of Wnt5a disrupts second heart field cell deployment and may contribute to OFT malformations in DiGeorge syndrome.</a> Human Molecular Genetics. 24 (6), 1704-1716 (2015).</li><li>Humphries, A. 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T., 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=Planar+cell+polarity+in+development+and+disease.">Planar cell polarity in development and disease.</a> Nature Reviews Molecular Cell Biology. 18 (6), 375-388 (2017).</li><li>Bradshaw, L., 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=Dual+role+for+neural+crest+cells+during+outflow+tract+septation+in+the+neural+crest-deficient+mutant+Splotch2H.">Dual role for neural crest cells during outflow tract septation in the neural crest-deficient mutant Splotch2H.</a> Journal of Anatomy. 214 (2), 245-257 (2009).</li><li>Kodo, K., 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=Regulation+of+Sema3c+and+the+interaction+between+cardiac+neural+crest+and+second+heart+field+during+outflow+tract+development.">Regulation of Sema3c and the interaction between cardiac neural crest and second heart field during outflow tract development.</a> Scientific Reports. 7 (1), 6771 (2017).</li><li>Waldo, K. L., 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=Cardiac+neural+crest+is+necessary+for+normal+addition+of+the+myocardium+to+the+arterial+pole+from+the+secondary+heart+field.">Cardiac neural crest is necessary for normal addition of the myocardium to the arterial pole from the secondary heart field.</a> Developmental Biology. 281 (1), 66-77 (2005).</li><li>Schleiffarth, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt5a+is+required+for+cardiac+outflow+tract+septation+in+mice.">Wnt5a is required for cardiac outflow tract septation in mice.</a> Pediatric Research. 61 (4), 386-391 (2007).</li><li>Nguyen, B. 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The noncanonical Wnt/planar cell polarity (PCP) signaling pathway is a critically important cellular signaling pathway that has been implicated in multiple developmental24,25 and disease processes24,26. During embryonic development, noncanonical Wnt signaling involves an expansive network of molecular signals from signal-sending cells that ultimately induce changes in morphology, asymmetric organ...

Noncanonical Wnt signaling is an evolutionarily conserved pathway that regulates cellular filament organization and directional migration. This pathway has been implicated in multiple biological processes, including embryonic tissue morphogenesis1,2,3, lymphatic and vascular angiogenesis4,5,6,7, and cancer growth and metastasis8,9,10. At the cellular level, noncanonical Wnt signaling is carried out through coordinated paracrine interactions between signal-sending and signal-receiving cells. These interactions frequently occur between cells of different lineages or types and involve a diverse molecular network that includes up to 19 ligands and multiple receptors, co-receptors, and downstream signal transduction effectors11. Further complicating this signaling process, previous studies have shown that ligand-receptor combinations can vary in a context- and tissue-dependent manner12,13, and that the same source ligands that drive noncanonical Wnt signaling in signal-receiving cells can be produced by multiple signal-sending cell types14,15. Given the cellular and molecular complexity associated with noncanonical Wnt signaling, the ability to study individual and clinically relevant mechanisms in vivo has been limited.
Attempts have been made to study noncanonical Wnt signaling using cell culture&#160;techniques in vitro. For example, wound-healing assays performed in cellular monolayers have been used to functionally assess cellular directional migration4,16,17,18,19. Immunostaining techniques have been used to perform spatial analyses of surface protein expression to evaluate noncanonical Wnt-induced changes in cellular morphology7,10, architecture, and asymmetric polarization18,19,20. Although these approaches have provided important tools for characterizing Wnt-related phenotypes in signal-receiving cells, the lack of signal-sending components in these protocols limits their ability to accurately model paracrine signaling mechanisms observed in vivo. As a result, there remains a critical need to develop in vitro systems that allow robust and reproducible evaluation of paracrine signaling interactions between signal-sending and receiving cells of the noncanonical Wnt pathway, particularly those of different cell types.
To this end, the primary objective of this study was to establish a protocol to model paracrine noncanonical Wnt signaling interactions in vitro. We developed a non-contact coculture system that recapitulates signal-sending and signal-receiving components of these interactions and allows the use of standard molecular, genetic, or pharmacologic approaches to independently study specific ligand-receptor mechanisms in the noncanonical Wnt pathway. Mechanisms of NCC-mediated Wnt signaling were examined in myoblasts using established murine cell lines. As proof of principle, this model was used to corroborate findings of prior in vivo studies in mice that implicate the Wnt5a-ROR2 axis as a relevant noncanonical Wnt signaling pathway between NCCs21 and SHF cardiomyoblast progenitors3,22,23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M-7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Antifade mounting medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200-10</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2323</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>C2C12 murine myoblast cell line</td>
			<td>ATCC</td>
			<td>CRL-1772</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks, 75 cm2</td>
			<td>ThermoFisher Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber Slide System, 4-well</td>
			<td>ThermoFisher Scientific</td>
			<td>154526</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM), high glucose (4.5 g/L), L-glutamine (2 mM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Falcon conical centrifuge tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon permeable support for 24-well plate with 0.4 &#181;M transparent PET membrane</td>
			<td>Corning</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>W3381E</td>
			<td>Stored in 50 mL aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Gelatin solution, 0.1%</td>
			<td>ATCC</td>
			<td>PCS-999-027</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Graduated and sterile pipette tips, 10 &#181;L</td>
			<td>USA Scientific</td>
			<td>1111-3810</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukemia inhibitory factor (LIF), 106 unit/mL</td>
			<td>Millipore Sigma</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778-075</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids (MEM NEAA) 100x</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Corning</td>
			<td>10-022-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Roche</td>
			<td>10107409001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-stick auto-glass coverslips, 24 x 55 mm</td>
			<td>Springside Scientific</td>
			<td>HRTCG2455</td>
			<td></td>
		</tr>
		<tr>
			<td>O9-1 neural crest cell line</td>
			<td>Millipore Sigma</td>
			<td>SCC049</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I, 1x</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution in PBS, 4%</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (10,000 U/mL penicillin and 10,000 &#956;g/mL streptomycin)</td>
			<td>Fisher Scientific</td>
			<td>W3470H</td>
			<td>Stored in 10 mL aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 488</td>
			<td>Abcam</td>
			<td>ab176753</td>
			<td>Stored at -20 &#176;C, Keep out of light</td>
		</tr>
		<tr>
			<td>Phosphate-buffer saline (PBS), 1x, without calcium and magnesium, pH 7.4</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant human fibroblast growth factor-basic (rhFGF-basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human/mouse Wnt5a protein</td>
			<td>R&#38;D Systems</td>
			<td>645-WN-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100 mM</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Square Petri dish with grid</td>
			<td>Thomas Scientific</td>
			<td>1219C98</td>
			<td></td>
		</tr>
		<tr>
			<td>STO murine fibroblast feeder cells</td>
			<td>ATCC</td>
			<td>CRL-1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 solution</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>Fisher Scientific</td>
			<td>W3513C</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Zeiss Apotome.2 fluoresence microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss inverted Axio Vert.A1 light microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen lite 2012 microscopy software</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>imaging software</td>
		</tr>
	</tbody>
</table>

modeling paracrine noncanonical wnt signaling in vitro

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches.
This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Effects of NCCs on migratory capacity of murine myoblasts 
This assay was first applied to evaluate the impact of NCCs on the migratory capacity of myoblasts. Figure 1 outlines the schematic model of the assay. To test this impact, scratch assays were performed with myoblasts that were grown in isolation (without NCC inserts) compared to those grown in the presence of inserts. As a positive control, 500 ng/mL of recombinant Wnt5a (rWnt5a) was added to chamber wells with...

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Modeling Paracrine Noncanonical Wnt Signaling In Vitro

The present study outlines a highly reproducible and tractable method to study paracrine noncanonical Wnt signaling events in vitro. This protocol was applied to evaluate the impact of paracrine Wnt5a signaling in murine neural crest cells and myoblasts.

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This ...

This protocol outlines a highly reproducible method to functionally and molecularly evaluate paracrine non-canonical Wnt signaling in any cell population of interest. Functional and molecular assessments are performed in the same cell population and can be applied to study any component of the Wnt/PCP pathway. To begin, resuspend the previously pelleted C2C12 cell in 10 milliliters of C2C12 medium.To dilute the cells at the ratio of 1:20, add 0.5 milliliters of cell suspension in a 15 milliliter tube containing 9.5 milliliters of fresh C2C12 medium and gently mix with a serological pipette using a P1000 pipette, add one milliliter of the diluted cell suspension to each well of a new four chambered well and place it in the incubator. For plating O9-1 cell inserts, resuspend the O9-1 cell pellet in 10 milliliters of O9-1 growth medium. And similar to C2C12 cell dilution, dilute the O9-1 cells at the ratio of 1:20.Next, place a single permeable insert inside each well of a new four chambered well filled with one milliliter of O9-1 growth medium. Add 300 microliters of the diluted O9-1 cell suspension to each insert and ensure that insert is submerged completely. Then incubate the well at 37 degrees Celsius.To perform siRNA knockdown in the O9-1 cells, dilute the siRNA and transfection reagent in the reduced serum medium according to the manufacturer's recommendations at the desired concentrations. Gently mix the diluted siRNA and transfection reagent and incubate the mixture at room temperature for seven minutes. After 18 to 24 hours of cell plating, add the siRNA lipid complexes to the O9-1 cell inserts and incubate for approximately 34 to 48 hours.When the cells reach 70 to 80%confluency, remove the medium from the C2C12 chamber well and wash the cells once with PBS. After removing the PBS, begin scratching the cells by passing a sterile P10 pipette tip firmly in a single direction to span the entire length or width of the cell monolayer. Once the cells are scratched, quickly add one milliliter of PBS.Next, turn on the computer and the microscope. Place the chamber slide on the stage and rotate the objective dial to 5X magnification. Open the imaging software.Click the camera tab and then click the Live button to visualize the cells on the AxioCam IC tab. Ensure that the light filter is pulled all the way out to allow light to pass to the camera and computer screen, manually move or rotate the chamber slide to position the wound area in the center of the live image on the AxioCam IC tab. To take images, click snap to open a new tab next to the AxioCam IC tab that contains the image.To save this still image, click on the file, select save as, select desktop on the bar on the left to save the file to the desktop, enter the file name in the file name box and save the figure in czi file format. To save the picture as a tiff, click file, select save as, enter the file name in the file name box. Click the save as type button and select tiff from the dropdown menu.Manually reposition the chamber slide to take two to three more images at other points of the wound in the same well. After imaging, remove the PBS from each well and add one milliliter of C2C12 medium. Assemble the well insert co-culture system by manually placing the inserts containing the O9-1 cells in each well of the chamber well.Gently push the inserts down into the well, such that the bottom of the insert sits just above the underlying C2C12 cells. Return the well insert constructs to the incubator and allow the cells to migrate for a total of nine to 12 hours. To determine the optimal migration time, check the cells at six hours following wound creation, then every two to three hours after that.End the experiment when the cells in control conditions completely cover the wound. Begin migration assay termination and well insert system deconstruction by removing the O9-1 cell inserts after nine to 12 hours of the migration period. Carefully aspirate the C2C12 medium.Add 0.5 milliliters of PBS to the chamber wells and take final images of cells following migration. Carefully aspirate all PBS mixed with medium and remove the plastic chambers from the chamber wells using kit instructions. Leaving the underlying slide containing the C2C12 cells.For immunostaining, immediately incubate the slide with 4%paraform aldehyde for 10 minutes at room temperature. Then remove paraformaldehyde and wash the slide with 0.1%Triton X-100 containing PBST for 15 minutes at room temperature. After 15 minutes, pour out PBST and wash the slide twice with PBS for 10 minutes each.Then create a hydrophobic boundary with a hydrophobic pen around the slide to prevent solutions from spilling from the slide preventing disruption of the adherence cells. Next, add approximately 0.5 milliliters of 1%BSA blocking solution to the slide within the hydrophobic boundary and incubate the slide for one hour at room temperature in a humidified slide chamber. At the end of the incubation, remove the blocking solution and add the phalloidin antibody to this slide within the hydrophobic boundary, and incubate at four degrees Celsius overnight.The next day, place the slide in a slide holder protected from light exposure. Wash the cells thrice with PBS for 10 minutes each at room temperature. Add DAPI-containing mounting medium, mount the slides with glass cover slips and capture the cells using a standard fluorescence microscope.In the present study, the addition of recombinant Wnt5a to co-culture wells accelerated myoblast migration with complete recovery of some areas by nine hours. The migratory myoblasts in all three conditions exhibited normal migratory cellular morphology, including well-formed and protruding filopodia and lamellipodia and asymmetric polarization of actin cytoskeletal projections. The paracrine effect of NCC-derived Wnt5a on myoblast migration was studied.Treatment with 50 nanomolar siRNA against Wnt5a reduced Wnt5a gene expression by 64%compared to negative control. The migration of C2C12 mile blasts with significantly reduced following Wnt5a knockdown in NCCs and the addition of exogenous recombinant Wnt5a rescued this migratory deficit and morphological defect in these myoblasts. In order to better understand the signal receiving cell mechanisms in the paracrine model, the ROR2 receptor was knocked down and gene expression was reduced by 54%Knockdown of ROR2 in myoblasts reduces their migratory capacity despite the presence of NCCs.Exogenous recombinant Wnt5a failed to rescue myoblast migration after ROR2 knockdown suggesting that ROR2 depletion disrupts the ability of myoblasts to receive Wnt5a signals. Culture cells to adequate density scratch with P10 tip only once assemble and unassembled co-culture system with care without disrupting underlying cell. This technique was used as a proof of concept to study a specific molecular signaling axis within the complex Wnt/PCP pathway with relevance for neural crest cell and second heart field biology.

modeling-paracrine-noncanonical-wnt-signaling-in-vitro

Research

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Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Analysis of Epididymal Protein Synthesis and Secretion

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation and storage of spermatozoa. Such complexity arises due to the combined secretory and absorptive activity of the lining epithelial cells. Here, we describe the techniques for the analysis of epididymal protein synthesis and secretion by focusing on the model protein family of dynamin (DNM) mechanoenzymes; large GTPases that have the potential to regulate bi-directional membrane trafficking events. For the study of protein expression in epididymal tissue, we describe robust methodology for immunofluorescence labeling of target proteins in paraffin-embedded sections and the subsequent detection of the spatial distribution of these proteins via immunofluorescence microscopy. We also describe optimized methodology for the isolation and characterization of exosome like vesicles, known as epididymosomes, which are secreted into the epididymal lumen to participate in intercellular communication with maturing sperm cells. As a complementary approach, we also describe the immunofluorescence detection of target proteins in an SV40-immortalized mouse caput epididymal epithelial (mECap18) cell line. Moreover, we discuss the utility of the mECap18 cell line as a suitable in vitro model with which to explore the regulation of epididymal secretory activity. For this purpose, we describe the culturing requirements for the maintenance of the mECap18 cell line and the use of selective pharmacological inhibition regimens that are capable of influencing their secretory protein profile. The latter are readily assessed via harvesting of conditioned culture medium, concentration of secreted proteins via trichloroacetic acid/acetone precipitation and their subsequent analysis via SDS-PAGE and immunoblotting. We contend that these combined methods are suitable for the analysis of alternative epididymal protein targets as a prelude to determining their functional role in sperm maturation and/or storage.

Here, we report the immunofluorescence localization of dynamin to illustrate the protocols for the detection of proteins in paraffin-embedded mouse epididymal sections and those of an immortalized epididymal cell line (mECap18). We also describe the protocols for the isolation of secretory proteins from both epididymal fluid and conditioned cell media.

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that na&#239;ve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- &#946;, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (&gt;70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (&gt;96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

A protocol outlining the formation of human blastoids that efficiently, timely, and sequentially generate blastocyst-like cells.

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend ...