Name: analysis of pluripotent stem cells by using cryosections of embryoid bodies
Uploaded: 2010-12-08T00:00:00
Description: Watch this Scientific Journal Video about Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies at JoVE.com

Dit werk werd ondersteund door Fundação de Amparo een Geavanceerd do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) en Instituto Nacional de Ciência e Tecnologia (INCTC). We zijn dankbaar dat Bruna S. Paulsen en Aline M. Fernandes voor de EB beelden.

1. Fixatie en Cryopreservatie Pluripotente stamcellen werden gekweekt op muis embryonale fibroblasten (MEF) geïnactiveerd met mitomycine C en onderhouden DMEM/F12 aangevuld met 20% knock-out serum vervanging (KSR) en 8ng / mL van fibroblast groeifactor (FGF-2). Met het oog op de vorming EB veroorzaken H9 cellen werden overgebracht naar niet-klevende gerechten en gekweekt gedurende 7 dagen, gehandhaafd in DMEM/F12 aangevuld met 15% KSR 8. Let op (!): Deze techniek kan g...

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	<li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-117 (1998).</li><li>Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., Benvenisty, N. <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, 88-95 (2000).</li><li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+inducing+embryoid+body+formation:+in+vitro+differentiation+system+of+embryonic+stem+cells.">Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells.</a> J Biosci Bioeng. 103, 389-398 (2007).</li><li>Conley, B. J., Young, J. C., Trounson, A. O., Mollard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+propagation+and+differentiation+of+human+embryonic+stem+cells.">Derivation propagation and differentiation of human embryonic stem cells.</a> Int J Biochem Cell Biol. 36, 555-567 (2004).</li><li>Moon, I. S., Cho, S. J., Jin, I., Walikonis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+combined+fluorescence+in+situ+hybridization+and+immunocytochemistry.">A simple method for combined fluorescence in situ hybridization and immunocytochemistry.</a> Mol Cells. 24, 76-82 (2007).</li><li>Daneshtalab, N., Dor&#233;, J. J., Smeda, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+tissue+specificity+and+antibody+selection:+Procedures+in+immunohistochemical+studies.">Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.</a> J Pharmacol Toxicol Methods. 61, 127-135 (2009).</li><li>Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval+for+immunohistochemical+reactions+in+routinely+processed+paraffin+sections.">Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections.</a> Methods Mol Biol. , 588-5103 (2010).</li><li>Nones, J., Spohr, T. C., Furtado, D. R., Sartore, R. C., Paulsen, B. S., Guimar&#227;es, M. Z., Rehen, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+cell+survival+in+embryoid+bodies.">Cannabinoids modulate cell survival in embryoid bodies.</a> Cell Biol Int. 12, 399-408 (2010).</li><li>Huang, W. M., Gibson, S. J., Facer, P., Gu, J., Polak, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+section+adhesion+for+immunocytochemistry+using+high+molecular+weight+polymers+of+L-lysine+as+a+slide+coating.">Improved section adhesion for immunocytochemistry using high molecular weight polymers of L-lysine as a slide coating.</a> Histochemistry. 77, 275-279 (1983).</li><li>Kuenzi, M. J., Sherwood, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+specific+relaxin-binding+cells+in+thecervix,+mammary+glands,+and+nipples+of+pregnant+rats.">Immunohistochemical localization of specific relaxin-binding cells in thecervix, mammary glands, and nipples of pregnant rats.</a> Endocrinology. 136, 1367-1373 (1995).</li></ol>

De hier beschreven methode biedt een eenvoudig te volgen protocol om PFA vaste dunne cryostaatcoupes van embryoid lichamen nuttig voor immunofluorescentie en in situ hybridisatie assays te verkrijgen. De resulterende vriescoupes toestaan ​​dat de studie van de cellulaire en moleculaire aspecten van menselijke embryonale stamcellen differentiatie, met behoud van hun structuur en organisatie als aggregaten.

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analysis of pluripotent stem cells by using cryosections of embryoid bodies

Embryonale stamcellen (ES)-cellen zijn pluripotent cellen, afkomstig van de binnenste celmassa van de blastocyst-stadium vroege zoogdieren embryo&#39;s 1. Een cruciale fase in de differentiatie van embryonale stamcellen is de vorming van embryoid organen (EBS) aggregaten 2, 3. EB vorming is gebaseerd op spontane aggregatie bij ES-cellen worden gekweekt in niet-aanhanger platen. Drie-dimensionale EB recapituleert vele aspecten van de vroege zoogdieren embryogenese en differentiëren tot de drie kiembladen: ectoderm, mesoderm en endoderm 4. Immunofluorescentie en in situ hybridisatie worden op grote schaal gebruikt technieken voor de detectie van target eiwitten en mRNA in cellen van een weefsel sectie 5, 6, 7. Hier presenteren wij een eenvoudige techniek om een ​​hoge kwaliteit vriescoupes van embryoid lichamen te genereren. Deze aanpak is gebaseerd op de ruimtelijke oriëntatie van de EB inbedding in oktober gevolgd door de cryosection techniek. De resulterende secties kunnen worden onderworpen aan een breed scala aan analytische procedures om populaties van cellen die bepaalde eiwitten, RNA of DNA te karakteriseren. In die zin is de voorbereiding van de EB vriescoupes (10μm) zijn essentiële hulpmiddelen voor histologie vlekken analyse (bijvoorbeeld Hematoxilin en eosine, DAPI), immunofluorescentie (bijv. Oct4, Nestin) of in situ hybridisatie. Deze techniek kan ook helpen om aspecten van de embryogenese met betrekking begrijpen het onderhoud van de driedimensionale bolvormige structuur van de EBS.

Watch this Scientific Journal Video about Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies at JoVE.com

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Pluripotente stamcellen in suspensie te groeien differentiëren in embryoid organen (EBS). Hier laten we zien hoe een hoge kwaliteit EB vriescoupes handig voor het bestuderen van cellulaire en moleculaire aspecten van de embryogenese te verkrijgen, met behoud van hun organisatie als aggregaten.

Analyse van de pluripotente stamcellen door gebruik te maken van vriescoupes Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

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