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

Detta arbete stöddes av Fundação de Amparo en Pesquisa gör Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) och Instituto Nacional de Ciencia e Tecnologia (INCTC). Vi är tacksamma för Bruna S. Paulsen och Aline M. Fernandes för EB bilder.

1. Fixering och Frysförvaring Pluripotenta stamceller odlades på möss embryonala fibroblaster (MEF) inaktiveras med mitomycin C och underhållas i DMEM/F12 kompletterad med 20% knockout serum ersättare (KSR) och 8ng / mL fibroblast tillväxtfaktor (FGF-2). För att förmå EB bildandet H9 celler överfördes till icke-häftande rätter och odlade i 7 dagar, upprätthålls i DMEM/F12 kompletterad med 15% KSR 8. Anmärkning (!): Denna teknik kan användas för EBS hä...

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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>

Den metod som beskrivs här ger en enkel att följa protokollet för att få PFA fast tunna kryostatsnitt av embryoid organ användbar för immunofluorescens och in situ hybridisering analyser. Den resulterande kryosnitt tillåter studier av cellulära och molekylära aspekter av mänskliga embryonala stamceller differentiering, samtidigt som deras struktur och organisation som aggregat.

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analysis of pluripotent stem cells by using cryosections of embryoid bodies

Embryonala stamceller (ES-celler) är pluripotenta celler som härrör från den inre cellmassan i blastocysten steg tidiga däggdjur embryon 1. En avgörande fas i differentiering av ES-celler är bildandet av embryoid organ (EBS) aggregat 2, 3. EB formation är baserad på spontana aggregering när ES-celler odlas i icke vidhäftande plattor. Tredimensionella EB rekapitulerar många aspekter av tidiga däggdjur embryogenesen och differentierar till de tre groddblad: ektoderm, mesoderm och endoderm 4. Immunofluorescens och in situ hybridisering används ofta tekniker för att upptäcka målproteiner och mRNA som finns i celler i en vävnad avsnitt 5, 6, 7. Här presenterar vi en enkel teknik för att generera hög kvalitet kryosnitt av embryoid organ. Detta synsätt bygger på rumslig orientering EB ingjutning i oktober följt av cryosection teknik. Den resulterande delarna kan utsättas för en mängd olika analysmetoder för att karaktärisera populationer av celler som innehåller vissa proteiner, RNA eller DNA. I denna mening, beredning av EB kryosnitt (10μm) är viktiga verktyg för histologi färgning analys (t.ex. Hematoxilin och Eosin, DAPI), immunofluorescens (t.ex. Oct4, nestin) eller in situ hybridisering. Denna teknik kan också hjälpa till att förstå aspekter av embryogenesen med avseende på underhållet av det tredimensionella sfäriska struktur 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

Pluripotenta stamceller växer i suspension differentieras till embryoid organ (EBS). Här visar vi hur du får hög kryosnitt kvalitet EB användbart för att studera cellulära och molekylära aspekter av embryogenes, samtidigt som deras organisation som aggregat.

Analys av pluripotenta stamceller genom att använda kryosnitt av Embryoid organ

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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