This study was supported by NIH grant R01 HL119984 to A.H.

1. Culture of Mouse Embryonic Fibroblasts (MEFs)
<ol>
	<li>Prepare MEF medium, Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, high-glucose), supplemented with 15% fetal bovine serum (FBS).</li>
	<li>Coat 100 mm cell culture dishes with 0.5% gelatin solution for 30 min at room temperature (RT).</li>
	<li>Count MEFs using a cytometer. Remove the gelatin solution and immediately pour MEF medium pre-warmed to 37 &#176;C. Rapidly thaw vials of mitomycin C-treated MEFs in a 37 &#176;C water bath for 2 min, then seed 2.8 x...

<ol>
	<li>Hopfl, G., Gassmann, M., Desbaillets, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+embryonic+stem+cells+into+embryoid+bodies.">Differentiating embryonic stem cells into embryoid bodies.</a> Methods Mol Biol. 254, 79-98 (2004).</li><li>Itskovitz-Eldor, J., 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=Differentiation+of+human+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Mol Med. 6 (2), 88-95 (2000).</li><li>Dang, S. M., Kyba, M., Perlingeiro, R., Daley, G. Q., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+embryoid+body+formation+and+hematopoietic+development+from+embryonic+stem+cells+in+different+culture+systems.">Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems.</a> Biotechnol Bioeng. 78 (4), 442-453 (2002).</li><li>Johansson, S., Svineng, G., Wennerberg, K., Armulik, A., Lohikangas, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-integrin+interactions.">Fibronectin-integrin interactions.</a> Front Biosci. 2, d126-d146 (1997).</li><li>Blumberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+retinoid+signaling+in+anteroposterior+neural+specification+and+neuronal+differentiation.">An essential role for retinoid signaling in anteroposterior neural specification and neuronal differentiation.</a> Semin Cell Dev Biol. 8 (4), 417-428 (1997).</li><li>Ross, S. A., McCaffery, P. J., Drager, U. C., De Luca, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+in+embryonal+development.">Retinoids in embryonal development.</a> Physiol Rev. 80 (3), 1021-1054 (2000).</li><li>Bain, G., Ray, W. J., Yao, M., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+promotes+neural+and+represses+mesodermal+gene+expression+in+mouse+embryonic+stem+cells+in+culture.">Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture.</a> Biochem Biophys Res Commun. 223 (3), 691-694 (1996).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Dev Biol. 275 (1), 124-142 (2004).</li><li>Duester, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+synthesis+and+signaling+during+early+organogenesis.">Retinoic acid synthesis and signaling during early organogenesis.</a> Cell. 134 (6), 921-931 (2008).</li><li>Niederreither, K., Dolle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+in+development:+towards+an+integrated+view.">Retinoic acid in development: towards an integrated view.</a> Nat Rev Genet. 9 (7), 541-553 (2008).</li><li>Maden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+in+the+development,+regeneration+and+maintenance+of+the+nervous+system.">Retinoic acid in the development, regeneration and maintenance of the nervous system.</a> Nat Rev Neurosci. 8 (10), 755-765 (2007).</li><li>Sheng, 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=Retinoic+acid+regulates+bone+morphogenic+protein+signal+duration+by+promoting+the+degradation+of+phosphorylated+Smad1.">Retinoic acid regulates bone morphogenic protein signal duration by promoting the degradation of phosphorylated Smad1.</a> Proc Natl Acad Sci U S A. 107 (44), 18886-18891 (2010).</li><li>Gajovic, S., St-Onge, L., Yokota, Y., Gruss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+mediates+Pax6+expression+during+in+vitro+differentiation+of+embryonic+stem+cells.">Retinoic acid mediates Pax6 expression during in vitro differentiation of embryonic stem cells.</a> Differentiation. 62 (4), 187-192 (1997).</li><li>Dong, D., Ruuska, S. E., Levinthal, D. J., Noy, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+cellular+retinoic+acid-binding+proteins+I+and+II+in+regulating+signaling+by+retinoic+acid.">Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid.</a> J Biol Chem. 274 (34), 23695-23698 (1999).</li><li>Sessler, R. J., Noy, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ligand-activated+nuclear+localization+signal+in+cellular+retinoic+acid+binding+protein-II.">A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II.</a> Mol Cell. 18 (3), 343-353 (2005).</li><li>Tang, S., 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=SIRT1-Mediated+Deacetylation+of+CRABPII+Regulates+Cellular+Retinoic+Acid+Signaling+and+Modulates+Embryonic+Stem+Cell+Differentiation.">SIRT1-Mediated Deacetylation of CRABPII Regulates Cellular Retinoic Acid Signaling and Modulates Embryonic Stem Cell Differentiation.</a> Mol Cell. 55 (6), 843-855 (2014).</li><li>Yang, J., 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=RhoA+inhibits+neural+differentiation+in+murine+stem+cells+through+multiple+mechanisms.">RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms.</a> Sci Signal. 9 (438), ra76 (2016).</li><li>Garnaas, M. 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=Syx,+a+RhoA+guanine+exchange+factor,+is+essential+for+angiogenesis+in+Vivo.">Syx, a RhoA guanine exchange factor, is essential for angiogenesis in Vivo.</a> Circ Res. 103 (7), 710-716 (2008).</li><li>Chou, Y. H., Khuon, S., Herrmann, H., Goldman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nestin+promotes+the+phosphorylation-dependent+disassembly+of+vimentin+intermediate+filaments+during+mitosis.">Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis.</a> Mol Biol Cell. 14 (4), 1468-1478 (2003).</li><li>Arai, T., Matsumoto, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+functionally+differentiated+microtubules+in+squid+neurons:+regional+distribution+of+microtubule-associated+proteins+and+beta-tubulin+isotypes.">Subcellular localization of functionally differentiated microtubules in squid neurons: regional distribution of microtubule-associated proteins and beta-tubulin isotypes.</a> J Neurochem. 51 (6), 1825-1838 (1988).</li><li>Arnhold, S., Klein, H., Semkova, I., Addicks, K., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurally+selected+embryonic+stem+cells+induce+tumor+formation+after+long-term+survival+following+engraftment+into+the+subretinal+space.">Neurally selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space.</a> Invest Ophthalmol Vis Sci. 45 (12), 4251-4255 (2004).</li><li>Liu, Y., 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=Retinoic+acid+receptor+beta+mediates+the+growth-inhibitory+effect+of+retinoic+acid+by+promoting+apoptosis+in+human+breast+cancer+cells.">Retinoic acid receptor beta mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells.</a> Mol Cell Biol. 16 (3), 1138-1149 (1996).</li><li>Altucci, 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=Retinoic+acid-induced+apoptosis+in+leukemia+cells+is+mediated+by+paracrine+action+of+tumor-selective+death+ligand+TRAIL.">Retinoic acid-induced apoptosis in leukemia cells is mediated by paracrine action of tumor-selective death ligand TRAIL.</a> Nat Med. 7 (6), 680-686 (2001).</li><li>Pettersson, F., Dalgleish, A. G., Bissonnette, R. P., Colston, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+cause+apoptosis+in+pancreatic+cancer+cells+via+activation+of+RAR-gamma+and+altered+expression+of+Bcl-2/Bax.">Retinoids cause apoptosis in pancreatic cancer cells via activation of RAR-gamma and altered expression of Bcl-2/Bax.</a> Br J Cancer. 87 (5), 555-561 (2002).</li><li>Kothapalli, C. R., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+matrix+microenvironment+for+targeted+differentiation+of+embryonic+stem+cells+into+neural+and+glial+lineages.">3D matrix microenvironment for targeted differentiation of embryonic stem cells into neural and glial lineages.</a> Biomaterials. 34 (25), 5995-6007 (2013).</li><li>Cai, J., 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=BMP+and+TGF-beta+pathway+mediators+are+critical+upstream+regulators+of+Wnt+signaling+during+midbrain+dopamine+differentiation+in+human+pluripotent+stem+cells.">BMP and TGF-beta pathway mediators are critical upstream regulators of Wnt signaling during midbrain dopamine differentiation in human pluripotent stem cells.</a> Dev Biol. 376 (1), 62-73 (2013).</li></ol>

The authors declare they have no competing or financial interests

In this protocol we present a relatively simple and accessible method to study neural differentiation of murine ESCs. In previous protocols, RA was added to the medium at day 2 or day 4 of the EB hanging-drop8 or by suspension culture7, respectively, or immediately after the EB hanging drop aggregation21. In the protocol we devised, RA was added earlier. Despite the earlier introduction of RA to EBs formed by suspension culture, this protocol produce...

ESCs originate from the blastocyst inner cell mass. These cells are pluripotent, i.e. they have the capacity to differentiate into any cell type of the organism of origin. ESC in vitro differentiation is of wide interest as an experimental system for investigating developmental pathways and mechanisms. It offers a potent and flexible model system to test new therapeutic approaches for correction of cell and tissue dysfunction. EBs recapitulate many aspects of cell differentiation during early embryogenesis. In particular, EBs can be used when embryonic lethality makes it difficult to determine the cellular basis of the embryonic defects1,2. EBs can be formed either by the hanging drop or liquid suspension techniques3. The advantage of the former is the ability to generate EBs of consistent size and density, thus facilitating experimental reproducibility.
Interaction with extracellular matrix (ECM) adhesion proteins may affect the motility and survival of adherent cells. In the 2D culture system, fibronectin is often applied to increase cell adhesion to the substrate. Fibronectin is a basal lamina component recognized by 10 types of cell-surface integrin heterodimers4.
RA is a small lipophilic metabolite of vitamin A that induces neural differentiation5,6. High concentrations of RA promote neural gene expression and represses mesodermal gene expression during EB formation7,8. RA is produced by vitamin A oxidation to retinaldehyde by either alcohol or retinol dehydrogenase, followed by retinaldehyde oxidation to the final product by retinaldehyde dehydrogenase9. Neural differentiation requires transport of RA from the cytoplasm to the nucleus by cellular RA-binding protein 2 (CRABP2). In the nucleus, RA binds to its cognate receptor complex consisting of a RAR-RXR heterodimer10. This results in recruitment of transcriptional co-activators, and the initiation of transcription9,11. Furthermore, RA promotes the degradation of phosphorylated (active) SMAD1, thus antagonizing BMP and SMAD signaling12. In addition to these activities, RA increases Pax6 expression, a transcription factor that supports neural differentiation13. RA signaling is modulated by sirtuin-1 (Sirt1), a nuclear nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that deacetylates CRABP2, interfering with its translocation to the nucleus, and hence with RA binding to the RAR-RXR heterodimer14,15,16.
Our goal in designing the RA-treated EB protocol described here is to optimize neural differentiation in order to facilitate in vitro analysis of the signaling pathways that regulate ESC differentiation into neuronal precursor cells. One of the advantages of this protocol is facilitation of the analysis of cell function by immunofluorescence. 3D EBs are not well penetrated by antibodies and are difficult to image. EB dissociation into a 2D monolayer at specific time points during neural differentiation facilitates immunolabeling and imaging of the cells by confocal microscopy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Materials</td>
		</tr>
		<tr>
			<td>MEFs</td>
			<td>EMD Millipore</td>
			<td>PMEF-CF</td>
			<td>ESC feeder layer</td>
		</tr>
		<tr>
			<td>ESC</td>
			<td>EMD Millipore</td>
			<td>CMTI-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (60 mm)</td>
			<td>Eppendorf</td>
			<td>30701119</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Cell culture dish (100 mm)</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Petri dish (100 mm)</td>
			<td>Corning</td>
			<td>351029</td>
			<td>Hanging drops</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662160</td>
			<td>2D EBs</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Eppendorf</td>
			<td>30720113</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>Dark 1.5 ml centrifuge tube</td>
			<td>Celltreat Scientific Products</td>
			<td>229437</td>
			<td>RA stock solution</td>
		</tr>
		<tr>
			<td>Microscope cover-glass</td>
			<td>Fisherbrand</td>
			<td>12-545-80</td>
			<td>Circular, 12 mm diameter</td>
		</tr>
		<tr>
			<td>Superfrost-plus microscope slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>3D collagen culture kit</td>
			<td>EMD Millipore</td>
			<td>ECM675</td>
			<td>3D culture</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen</td>
			<td>301427</td>
			<td>Stem cell transfection</td>
		</tr>
		<tr>
			<td>Microcon&#160;Centrifugal Filters (10 kDa)</td>
			<td>EMD Millipore</td>
			<td>MRCPRT010</td>
			<td>Protein concentration</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Reagents</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>12-709F</td>
			<td>MEFs culture</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>ESCs culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>EMD Millipore</td>
			<td>ES-009-B</td>
			<td>ESCs culture</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G2625</td>
			<td>Dish coating</td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>R&#38;D Systems</td>
			<td>8878-LF-025</td>
			<td>To maintain ESC pluripotency</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solutions</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750060</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>MycoZap Plus-PR</td>
			<td>Lonza</td>
			<td>VZA-2022</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans-retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-50MG</td>
			<td>Induction of neural differentiation</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-50G</td>
			<td>Blocking and antibody dilution&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-100ML</td>
			<td>Cell membrane permeabilization</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold anti-fade reagent with DAPI</td>
			<td>Life Tech.</td>
			<td>P36931</td>
			<td>Mounting reagent</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Cell fixation</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>R&#38;D Systems</td>
			<td>1030-FN</td>
			<td>Dish coating</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Worthington Biochem. Corp</td>
			<td>LS004196</td>
			<td>EB dissociation</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Primary Antibodies</td>
		</tr>
		<tr>
			<td>Nestin (Rat-401)</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-33677</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Oct4</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-5279</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Nanog</td>
			<td>Bethyl Laboratories</td>
			<td>A300-398A</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Sox2</td>
			<td>Cell Signaling</td>
			<td>3579</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Tubulin b3 (AA10)</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-80016</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Secondary Antibodies</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse-Alexa555</td>
			<td>Life Tech.</td>
			<td>A31570</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse-Alexa488&#160;</td>
			<td>Life Tech.</td>
			<td>A21202</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Instruments</td>
		</tr>
		<tr>
			<td>Wide-field microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>Cell culture imaging</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>C2</td>
			<td>Immunofluorescence imaging</td>
		</tr>
	</tbody>
</table>

analysis of retinoic acid-induced neural differentiation of mouse embryonic stem cells in two and three-dimensional embryoid bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

Oct4, Nanog, and SOX2 are the core transcription factors that confer ESC self-renewal and pluripotency. We applied the above protocol to compare the neural differentiation of ESCs from wild type and from a strain of genetically-modified mice where Syx, a gene coding for the RhoA-specific exchange factor Syx, is disrupted. We had implicated Syx in angiogenesis18. We noticed differences in the behaviors of EBs aggregated from Syx+/+ and <...

Watch this Scientific Journal Video about Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies at JoVE.com

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

The overall goal of this procedure is to analyze the expression and production of genes and protein markers, respectively, of neural differentiation of mouse embryonic stem cells. This method can help determine the mechanism responsible for the inhibition of neural differentiation by the raw age of DPAs, and lead to better protocols to differentiate stem cells into neurons for therapeutic purposes. The main advantage of this technique is that it facilitates tracking the process of neural differentiation at the emerin and protein levels, including by optical microscopy.Demonstrating the procedure will be Dr.Junning Yang, a research associate in my laboratory. Begin embryoid body formation by harvesting embryonic stem cells with 0.25%Trypsin-EDTA for two to five minutes at 37 degrees celsius, 5%carbon dioxide. Add fresh Iscove's Modified Dulbecco's Medium, or IMDM, with 15%FBS to the detaching cells, and transfer the cell suspension to a 15 milliliter tube.Centrifuge at 160 x G for five minutes at room temperature. Then count the cells using a hemocytometer, and prepare a five x 10 to the fifth cells per milliliter suspension in IMDM with 0.5 micromolar retinoic acid. Next, use an eight-channel pipette and 200-microliter tips to plate 120-microliter drops per 100 millimeter petri dish.Invert the dish and fill the inverted lid with PBS to prevent hanging drops from drying. Culture at 37 degrees celsius, 5%carbon dioxide, for three to four days. Use 200-microliter pipette tips to harvest hanging drop embryoid bodies grown for three days.Touch an embryoid body with a pipette tip, and transfer it to a fibernectin-coated cover slip in a 24-well plate. Change the medium on the cultures every two to three days. When changing the medium, collect the used medium for protein secretion analysis as needed.To detect secreted proteins in the medium, transfer 500 microliters of embryoid body medium to 10 kilodalton cutoff centrifugal filters, and spin at 20, 000 x G at four degrees celsius. Examine the medium remaining inside the centrifugal filters every 15 to 20 minutes to prevent excessive enrichment. Stop centrifugation when the volume of the remaining medium has fallen to 25 microliters.Invert the filter and spin at 160 x G at four degrees celsius, then collect the remaining concentrated medium by following the manufacturer's instructions. For a 3D culture, harvest embryoid bodies grown for four days as hanging drops with 200-microliter pipette tips, as before. Transfer 30 embryoid bodies to a 1.5 milliliter centrifuge tube, then prepare collagen gel solution by diluting appropriate volumes of collagen solution and 5X DMEM, adding the neutralization solution, and immediately mixing well.Place the collagen gel on ice. Next, pipette an appropriate volume of chilled collagen solution into the tube containing the embryoid bodies, and then gently pipette the embryoid body suspension into the wells of a six-well plate without creating bubbles. Immediately transfer the plate to 37 degrees celsius, 5%carbon dioxide, for 60 minutes to initiate collagen polymerization.After the 60 minutes, overlay the plate containing the embryoid bodies with IMDM. For embryoid body dissociation, again collect the four-day hanging drop embryoid bodies with 200-microliter pipette tips. This time, transfer them to a non-adhesive bacteriological petri dish containing IMDM with 15%FBS and culture at 37 degrees celsius, 5%carbon dioxide, for four more days.Check the embryoid bodies twice every day to make sure they do not attach to the bottom. Gently shake the dish to prevent embryoid body attachment. After four days, transfer the embryoid bodies to a centrifuge tube and centrifuge at 185 x G for five minutes at room temperature.Following centrifugation, remove the supernatant. The palette volume should not exceed 100 microliters. Next, pipette one milliliter of 0.25%type I collagenase, supplemented with 20%FBS in PBS into the tube of embryoid bodies.Incubate the mixture for one hour at 37 degrees celsius and 5%carbon dioxide. Gently pipette the suspension using a one-milliliter pipette tip every 20 minutes. After the incubation, gently wash the cells three times with PBS.If cell aggregates are present, use a cell strainer with a 100-micron mesh to remove them. Plate the cells on gelatin-coated cover slips in IMDM, then incubate at 37 degrees celsius and 5%carbon dioxide. Wash the 2D or dissociated 3D embryoid bodies with PBS.Then fix with four percent paraformaldehyde and PBS for 30 minutes at room temperature. After washing the fixed cells three times with PBS to remove floating cell debris, add 1%triton X-100 in PBS to permeabilize the cells for 20 minutes at room temperature. After washing as before, remove the last wash and block with 5%BSA in PBS for 30 minutes.Towards the end of the incubation, prepare a primary antibody dillution in 1%BSA in PBS supplemented with 0.03%triton X-100. When the 30 minutes has elapsed, replace the blocking solution with the primary antibody solution. Incubate for three hours at room temperature, or overnight, at four degrees celsius.After washing, apply a secondary antibody in PBS and incubate for one hour at room temperature. Finally, mount cover slips on glass slides with an anti-fade medium for optical microscopy. This image shows sprout emergence from an embryoid body generated from ESCs, positive for two copies of the Syx gene, which codes for a guanine exchange factor that activates the GTPase RhoA.The image was taken after six days in 3D culture. This embryoid body formed from Syx KnockOut ESCs demonstrates increased sprouting after the same time in culture. Here, phase images of 2D embryoid body edges show that cells expanded faster from Syx KnockOuts than from Syx wild-type.These immunofluorescence images illustrate that the neural differentiation marker, Nestin, shown in red, was more abundant in cells expanding from six-day 2D6 KnockOut embryoid bodies than from their Syx wild-type counterparts. Here, immunofluorescence images show that the neural differentiation markers Nestin and beta III Tubulin were more abundant in cells dissociated from 13-day 3D6 KnockOut embryoid bodies than from their Syx wild-type counterparts. This is a time-consuming procedure because of the duration of embryoid body growth and differentiation.It not only requires at least eight days, but in some cases we follow differentiation up to 24 days. While attempting this procedure, it is important to remember to limit the viability of embryoid body diameter to ensure similar differentiation rates along the embryoid bodies.

analysis-retinoic-acid-induced-neural-differentiation-mouse-embryonic

Research

JoVE Journal

Developmental Biology

8.8K Views.  Sidney Kimmel Medical College, Thomas Jefferson University. The overall goal of this procedure is to analyze the expression and production of genes and protein markers, respectively, of neural differentiation of mouse embryonic stem cells. This method can help determine the mechanism responsible for the inhibition of neural differentiation by the raw age of DPAs, and lead to better protocols to differentiate stem cells into neurons for therapeutic purposes. The main advantage of this technique is that it facilitates tracking the process of neural diff...