Name: epigenetic regulation of cardiac differentiation of embryonic stem cells and tissues
Uploaded: 2016-06-03T14:00:00
Description: Watch this Scientific Journal Video about Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues at JoVE.com

The authors acknowledge funding agencies, the IMI StemBANCC European community programme, the leducq Foundation (SHAPEHEART) and the Agence Nationale de la Recherche (Genopath)

1. DNA-protein Tværbinding <ol><li> Fix i 15 ml rør høstet-ES-celler (2 x 10 6 celler til regelmæssig chip, 2 x 10 5 celler til mikrochip), embryoide organer (EBS), der genereres fra ES-celler og embryonale hjerte væv dissekeret fra E9.5 musefostre (AV kanalen, udstrømning tarmkanalen og ventrikel) under anvendelse af 1% formaldehyd i PBS i celler eller i permeabilisering PB2 buffer for embryonale væv. Anbring glassene på orbitalryster ved en hastighed på 60 rpm ved stuetemperatur i nøj...

<ol>
	<li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+mammalian+heart+from+two+sources+of+myocardial+cells.">Building the mammalian heart from two sources of myocardial cells.</a> Nat Rev Genet. 6, 826-835 (2005).</li><li>Chen, T., Dent, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifiers+and+remodellers:+regulators+of+cellular+differentiation.">Chromatin modifiers and remodellers: regulators of cellular differentiation.</a> Nat Rev Genet. 15, 93-106 (2014).</li><li>Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+state+of+chromatin+immunoprecipitation.">The current state of chromatin immunoprecipitation.</a> Mol Biotechnol. 45, 87-100 (2010).</li><li>Gade, P., Kalvakolanu, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+assay+as+a+tool+for+analyzing+transcription+factor+activity.">Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity.</a> Methods Mol Biol. 809, 85-104 (2012).</li><li>Scott, V., Clark, A. R., Docherty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+retardation+assay.">The gel retardation assay.</a> Methods Mol Biol. 31, 339-347 (1994).</li><li>Van Vliet, P., Wu, S. M., Zaffran, S., Puceat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+cardiac+development:+a+view+from+stem+cells+to+embryos.">Early cardiac development: a view from stem cells to embryos.</a> Cardiovasc Res. 96, 352-362 (2012).</li><li>Leschik, J., Caron, L., Yang, H., Cowan, C., Puceat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+of+bivalent+epigenetic+marks+in+two+human+embryonic+stem+cell+lines+reveals+a+different+cardiogenic+potential.">A view of bivalent epigenetic marks in two human embryonic stem cell lines reveals a different cardiogenic potential.</a> Stem Cells Dev. 24, 384-392 (2015).</li><li>Bonn, S., Zinzen, R. P., Girardot, C., Gustafson, E. H., Perez-Gonzalez, A., Delhomme, N., Ghavi-Helm, Y., Wilczynski, B., Riddell, A., Furlong, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+analysis+of+chromatin+state+identifies+temporal+signatures+of+enhancer+activity+during+embryonic+development.">Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development.</a> Nat Genet. 44, 148-156 (2012).</li><li>Kim, T. K., Shiekhattar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectural+and+Functional+Commonalities+between+Enhancers+and+Promoters.">Architectural and Functional Commonalities between Enhancers and Promoters.</a> Cell. 162, 948-959 (2015).</li><li>Abboud, N., Moore-Morris, T., Hiriart, E., Yang, H., Bezerra, H., Gualazzi, M. G., Stefanovic, S., Guenantin, A. C., Evans, S. M., Puceat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cohesin-OCT4+complex+mediates+Sox+enhancers+to+prime+an+early+embryonic+lineage.">A cohesin-OCT4 complex mediates Sox enhancers to prime an early embryonic lineage.</a> Nat Commun. 6, 6749 (2015).</li><li>Dahl, J. A., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Q2ChIP,+a+quick+and+quantitative+chromatin+immunoprecipitation+assay,+unravels+epigenetic+dynamics+of+developmentally+regulated+genes+in+human+carcinoma+cells.">Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells.</a> Stem Cells. 25, 1037-1046 (2007).</li><li>Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, 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=A+bivalent+chromatin+structure+marks+key+developmental+genes+in+embryonic+stem+cells.">A bivalent chromatin structure marks key developmental genes in embryonic stem cells.</a> Cell. 125, 315-326 (2006).</li><li>Wamstad, J. A., Alexander, J. M., Truty, R. M., Shrikumar, A., Li, F., Eilertson, K. E., Ding, H., Wylie, J. N., Pico, A. R., Capra, J. 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=Dynamic+and+coordinated+epigenetic+regulation+of+developmental+transitions+in+the+cardiac+lineage.">Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage.</a> Cell. 151, 206-220 (2012).</li><li>Stergachis, A. B., Neph, S., Reynolds, A., Humbert, R., Miller, B., Paige, S. L., Vernot, B., Cheng, J. B., Thurman, R. E., Sandstrom, 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=Developmental+fate+and+cellular+maturity+encoded+in+human+regulatory+DNA+landscapes.">Developmental fate and cellular maturity encoded in human regulatory DNA landscapes.</a> Cell. 154, 888-903 (2013).</li><li>Brind'Amour, J., Liu, S., Hudson, M., Chen, C., Karimi, M. M., Lorincz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-low-input+native+ChIP-seq+protocol+for+genome-wide+profiling+of+rare+cell+populations.">An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations.</a> Nat Commun. 6, 6033 (2015).</li></ol>

Epigenetik er blevet et vigtigt forskningsområde i udviklingsmæssige biologi. Hvordan et genetisk program aktiveres i embryonale celler for at give cellerne erhverve særlige identitet i en embryonal slægt har været i lang tid et centralt spørgsmål for udviklingsmæssige biologer. Chip har været bredt anvendt inden for de sidste år, og kombineres til DNA-sekventering efter forbedring i opløsning på sekventering. Dette er blevet en kraftfuld teknik for at undersøge i et genom bred ...

<html> Hjertet er det første organ, der skal dannes, og at blive funktionelle i embryoet. Hjertet er bygget af mange cellelinier, der opstår fra den første og anden embryonale hjerte felter 1. Fra post-befrugtning blastocyst-stadiet til formede hjerte, har embryonale celler således at gøre mange celle fate beslutninger. Gentransskription reguleres på en tids- og rum-afhængig måde og er en vigtig biologisk proces, der ligger til grund celleskæbne beslutning under fosterudviklingen. En sådan proces medieres af specifikke transkriptionsfaktorer, som binder regulatoriske regioner i genomet, herunder enhancere og promotorer af kardiale konstitutive gener. DNA er viklet rundt histoner, der udsættes for modifikationer, såsom acetylering, methylering, ubiquitinylation, og / eller phosphorylering. Histon modifikation fører til undertrykt, aktiveret eller poised gentransskription afhængigt af hvilken lysinrest af histon modificeres 2. jove_content &quot;&gt; chromatin immunopræcipitationsassay (chip) er blevet oprettet år siden 3 og er i øjeblikket den mest bredt anvendt teknologi for at identificere mål af enten modificerede histoner eller transkriptionsfaktorer fire. Efter immunopræcipitation af histoner eller transkriptionsfaktorer, kan bindes DNA være enten amplificeret ved polymerasekædereaktion (PCR) eller sekventeret. chip har teknisk overvundet mere udfordrende gel retarderingsassays 5. chip betyder imidlertid ikke, at direkte binding af en transkriptionsfaktor til DNA, en fordel ved gelforsinkelsesassay. på den anden side, ChIP kombineres til DNA-sekventering har åbnet en ny genom-dækkende perspektiv på genregulering. ES-celler (ES-celler) rekapitulere inden embryoide legemer (dvs.., Celleaggregater) eller i 2D kultur de tidlige trin i hjerte- udvikling 6 og tilvejebringer i princippet tilstrækkeligt materiale til chip. Endvidere humane ES-celler repræsenterer en human celle model af cardiogenesis selvom deres kardiogent potentiale afhænger af deres epigenetisk signatur 7. På senere stadier af udvikling, mus embryonale væv mulighed for at undersøge specifikke epigenetiske landskaber kræves til bestemmelse af celle identitet. Imidlertid er genomet transkriberes i en tids- og celletype-specifik måde 8. Epigenetisk regulering af gentranskription der skal læses inden lokaliserede regioner. Heri beskriver vi protokoller chip, sekventiel chip efterfulgt af PCR eller sekventering ved hjælp ES-celler, embryoide organer og hjerte-specifikke embryonale regioner. Disse protokoller gør det muligt at undersøge epigenetisk regulering af hjerte gentranskription.

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Formaldehyde&#160;</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:147px;">F8775</td>
			<td style="width:167px;">Cell Fixation&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td>Cross-link stop</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aprotinin</td>
			<td>Fluka</td>
			<td>10820</td>
			<td>Proteases inhibitor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Leupeptin hemisulfate</td>
			<td>Sigma</td>
			<td>L2882</td>
			<td>Proteases inhibitor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>Proteases inhibitor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Protein A magnetic beads&#160;</td>
			<td>Life technologies</td>
			<td>10001D</td>
			<td>Immunoprecipitation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">SPRI magnetic beads</td>
			<td>Thermo Scientific</td>
			<td>15002-01</td>
			<td>DNA purification</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Proteinase K</td>
			<td>Life technologies</td>
			<td>25530-015</td>
			<td>Protein digestion&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DNA BR standard&#160;</td>
			<td>Life technologies</td>
			<td>Q32850</td>
			<td>Calibration range&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Syber green</td>
			<td>Molecular Probes</td>
			<td>S-11484</td>
			<td>DNA quantification</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">TE buffer&#160;</td>
			<td>Invitrogen</td>
			<td>P7589</td>
			<td>DNA quantification</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PBX 1X</td>
			<td>Life technologies</td>
			<td>14190-094</td>
			<td>Washing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DNase RNase free water</td>
			<td>Life technologies</td>
			<td>10977-035</td>
			<td>Dilution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Axygen tube</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td>ChIP purifiction</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibody&#160;</td>
			<td>Company</td>
			<td>Reference</td>
			<td style="width:167px;">ChIP concentration</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K27ac</td>
			<td>Abcam</td>
			<td>ab4729</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K4me1</td>
			<td>Diagenode</td>
			<td>C15410194 (pAb-194-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K36me3</td>
			<td>Diagenode</td>
			<td>C15410058 (pAb-058-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K9me2</td>
			<td>Diagenode</td>
			<td>C15410060 (pAb-060-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K4me3</td>
			<td>Diagenode</td>
			<td>C15410030 (pAb-030-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K27me3</td>
			<td>Diagenode</td>
			<td>C15410069 (pAb-069-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
	</tbody>
</table>

epigenetic regulation of cardiac differentiation of embryonic stem cells and tissues

Specifik gentransskription er en vigtig biologisk proces, der ligger til grund celleskæbne beslutning under fosterudviklingen. Den biologiske proces medieres af transkriptionsfaktorer, som binder genomiske regulatoriske regioner, herunder forstærkere og initiativtagere til hjerte-konstitutive gener. DNA er viklet rundt histoner, der udsættes for kemiske modifikationer. Ændringer af histoner føre yderligere til undertrykt, aktiveret eller balancerer gentranskription, hvilket bringer et andet niveau af finjustering regulering af gen transskription. Embryonale stamceller (ES-celler) rekapitulere inden embryoide legemer (dvs. celleaggregater) eller i 2D kultur de tidlige trin i hjerte- udvikling. De giver i princippet nok materiale til kromatin immunofældning (chip), en teknologi bredt anvendt til at identificere regulatoriske regioner gen. Endvidere humane ES-celler repræsenterer en human celle model af cardiogenese. På senere stadier af udvikling, mus embryonale væv tilladerundersøge specifikke epigenetiske landskaber kræves til bestemmelse af celle identitet. Heri beskriver vi protokoller chip, sekventiel chip efterfulgt af PCR eller chip-sekventering ved hjælp ES-celler, embryoide organer og hjerte-specifikke embryonale regioner. Disse protokoller gør det muligt at undersøge epigenetisk regulering af hjerte gentranskription.

Figur 1A viser først fremstillingen af DNA-bindende perler og kvalitetskontrol under anvendelse af DNA fra forskellige størrelser (1 kb stige). 0ne, 2 og 2,5 voluminer (1 til 3) af perler blev tilsat til et volumen af ​​prøven til oprensning med høj og lav molekylær størrelse DNA-fragmenter. Figurerne 1 B, C, D er typiske eksempler på DNA-geler fra hele sonikeret DNA ekstraheret fra...

Watch this Scientific Journal Video about Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues at JoVE.com

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

En finjustering regulering af gen transskription ligger til grund embryonal celle skæbne beslutning. Heri beskriver vi chromatin immunopræcipitationsanalyser anvendt til at undersøge epigenetisk regulering af både hjerte- differentiering af stamceller og kardial udvikling af musefostre.

Epigenetisk forordning af Cardiac Differentiering af embryonale stamceller og væv

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

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Afledning af Cardiac progenitorceller af embryonale stamceller

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

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

Neural Differentiering af Mouse Embryonale stamceller i Serumfrit monolagskultur

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

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Isolering og differentiering af adipøst afledte stamceller fra Porcine subkutane fedtvæv

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

Isolering af murint embryonale Hemogenic endotelceller

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Storstilet produktion af cardiomyocytter fra menneskelige pluripotente stamceller Ved hjælp af en meget reproducerbar lille molekyle-Based Differentiering Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Samlet Size optimering i Microwells for suspension-baserede Cardiac Differentiering af menneskelige pluripotente stamceller

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

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

Analyse af retinoidsyre-induceret Neural Differentiation af muse embryonale stamceller i to og tre-dimensionelle embryoide legemer

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 ...

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

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente stamceller

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

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Identifikation af skeletmuskulatur satellit celler ved immunfluorescens med Pax7 og Laminin antistoffer

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

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

Beregningsmæssige analyse af Caenorhabditis elegans kønscelleoverførsel at studere Distribution kerner, proteiner og cytoskelettet

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