Name: analysis of retinoic acid-induced neural differentiation of mouse embryonic stem cells in two and three-dimensional embryoid bodies
Uploaded: 2017-04-22T14:00:00
Description: 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

这项研究是由美国国立卫生研究院授予R01 HL119984到AH支持

1.培养小鼠胚胎成纤维细胞的（MEF中） <ol><li>制备MEF培养基，Dulbecco改良的Eagle氏培养基（DMEM，高葡萄糖），补充有15％胎牛血清（FBS）。 </li><li>外套百毫米细胞培养皿以在室温下（RT）30分钟0.5％的明胶溶液。 </li><li>计数使用流式细胞仪的MEF。除去明胶溶液，并立即倒入MEF培养基预先加热至37℃。迅速解冻丝裂霉素C处理的MEF的小瓶在37℃水浴中2分钟，然后播种每100mm明胶包被的培养皿2.8×10 6...

<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>

在这个协议中，我们提出一个相对简单的和方便的方法来研究的鼠胚胎干细胞的神经分化。在先前的协议中，RA在第2天或EB悬滴8的第4天或由悬浮培养7加入到培养基中，分别或后立即EB悬滴聚集21。在我们设计的方案，RA较早加入。尽管较早引进RA的到的EB通过悬浮培养形成的，这个方案产生的神经分化标记8更高的表达。 <p c...

胚胎干细胞从胚泡内细胞团起源。这些细胞是多能的， 即它们具有分化成起源的生物体的任何细胞类型的能力。 ESC 体外分化为广泛兴趣，作为调查的发育途径和机制的实验系统。它提供了一个强有力的和灵活的模型系统来测试的细胞和组织功能障碍修正新的治疗方法。 EB的早期胚胎发育过程中重演细胞分化的许多方面。具体地，可以当胚胎致死性使得难以确定胚胎缺陷1,2的蜂窝式的基础上使用的EB。的EB可以通过悬滴或液体悬浮液技术3形成。前者的优点是，以产生一致的大小和密度的EB中，从而有利于实验的再现性的能力。 4识别的基底膜成分。 RA是诱导神经分化5,6维生素A的一小的亲脂性代谢物。高浓度的RA的促进神经基因表达和在EB形成7,8阻遏中胚层的基因表达。 RA是由维生素A的氧化通过任一醇或视黄醇脱氢酶，随后醛氧化成最终产品通过醛脱氢酶9醛生产。神经分化需要从细胞质RA的运输<html>由蜂窝RA-结合蛋白2（CRABP2）细胞核。在细胞核中，RA结合由一个RAR-RXR异二聚体10的其同源受体复合物。这导致招聘转录共激活因子和转录9,11的开始。此外，RA促进磷酸化的（活性）SMAD1的降解，从而拮抗BMP和Smad信号12。除了这些活动，RA增加Pax6的表达，支持神经分化13的转录因子。 RA信令由沉默调节蛋白1（Sirt1的），核烟酰胺腺嘌呤二核苷酸（NAD +）调制-即deacetylates CRABP2，以其易位干扰到细胞核依赖性酶，并因此与RA结合RAR-RXR异二聚体14,15 16。 e_content“&gt;我们在设计这里所述的RA处理的EB协议的目标是为了便于调节ESC分化成神经元前体细胞中的信号传导途径的体外分析，以优化神经分化，其中一个这种协议的优点之一是便利细胞功能的免疫荧光。3D的EB的分析不能很好地穿透抗体和难以成像。EB解离成在特定的时间点的2D单层期间神经分化通过共聚焦显微镜促进免疫标记和细胞的成像。

<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

从胚泡（通常在一天E3.5）的内质分离的小鼠胚胎干细胞（ESC），可以用作体外模型系统用于研究早期胚胎发育。在不存在白血病抑制因子（LIF）的，胚胎干细胞分化默认成神经前体细胞。它们可以积累成三维（3D）的球形聚集体称为胚状体（EB），由于其相似性的早期胚胎中。的EB可在纤连蛋白包被的盖玻片接种，在那里它们通过生长的二维（2D）扩展展开，或在3D胶原基质在那里继续成长为球状体，并分化成三个胚层注入：内胚层，中胚层和外胚层。三维胶原文化更加接近地模拟体内环境比2D日圆。所述2D EB培养通过免疫荧光和免疫印迹来跟踪分化促进分析。我们已经制定了两步神经不同点重刑协议。在第一步骤中，的EB通过悬滴技术生成的，并且，同时，被诱导通过暴露于视黄酸（RA）进行区分。在第二步骤中，在不存在RA的2D或3D格式神经分化前进。

OCT4，NANOG，SOX2和是赋予ESC自我更新和多能性的核心转录因子。我们应用上述协议ESC的神经分化从野生型和从那里SYX，一个基因编码的RhoA特定交换因子SYX，被破坏基因改造的小鼠的菌株进行比较。我们曾在18血管牵连三亚。我们注意到，在EB的行为差异从三亚+ / +和三亚汇总- / -胚胎干细胞，并且进行了测试，如?...

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

我们描述了使用小鼠胚胎干细胞生成二维或三维的胚状体的技术。然后，我们解释如何以诱导胚状体细胞视黄酸，以及如何的神经分化由祖细胞标志物的免疫荧光和免疫印迹来分析其分化状态。

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

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.

Research

JoVE Journal

Developmental Biology

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

从诱导多能干得出视网膜色素上皮（RPE）（IPS），由胚体大小不同的细胞

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

科研

发育生物学

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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

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

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

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

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

从原生多能诱导干细胞中生成胚胎体表面的人原始生殖细胞样细胞

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...