Name: pluripotent stem cell derived cardiac cells for myocardial repair
Uploaded: 2017-02-03T15:00:00
Description: Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).

Toutes les procédures expérimentales sont réalisées en conformité avec les lignes directrices animales de l&#39;Université d&#39;Alabama à Birmingham. 1. Différencier hiPSCs dans hiPSC-CMs <ol><li> Revêtir les puits d&#39;une plaque à 6 puits avec un mélange de protéines gélatineux prérefroidie aux facteurs de croissance réduite à 4 ° C pendant une nuit. Aspirer le mélange de protéines gélatineux avant utilisation. Ensemencer les hiPSCs sur les plaques pré-revêtues, e...

<ol>
	<li>Qiao, H., 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=Death+and+proliferation+time+course+of+stem+cells+transplanted+in+the+myocardium.">Death and proliferation time course of stem cells transplanted in the myocardium.</a> Mol Imaging Biol. 11 (6), 408-414 (2009).</li><li>Ye, 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=Cardiac+repair+in+a+porcine+model+of+acute+myocardial+infarction+with+human+induced+pluripotent+stem+cell-derived+cardiovascular+cells.">Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells.</a> Cell Stem Cell. 15 (6), 750-761 (2014).</li><li>Zhang, S., Dutton, J. R., Su, L., Zhang, J., Ye, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+a+spatiotemporal+3D+environment+on+endothelial+cell+differentiation+of+human+induced+pluripotent+stem+cells.">The influence of a spatiotemporal 3D environment on endothelial cell differentiation of human induced pluripotent stem cells.</a> Biomaterials. 35 (12), 3786-3793 (2014).</li><li>Yang, 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=Differentiation+of+Human+Induced-Pluripotent+Stem+Cells+into+Smooth-Muscle+Cells:+Two+Novel+Protocols.">Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols.</a> PLoS One. 11 (1), e0147155 (2016).</li><li>Rensen, S. S., Doevendans, P. A., van Eys, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+characteristics+of+vascular+smooth+muscle+cell+phenotypic+diversity.">Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.</a> Neth Heart J. 15 (3), 100-108 (2007).</li><li>Xu, C., Police, S., Hassanipour, M., Gold, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+bodies:+a+novel+culture+method+for+enrichment+of+cardiomyocytes+derived+from+human+embryonic+stem+cells.">Cardiac bodies: a novel culture method for enrichment of cardiomyocytes derived from human embryonic stem cells.</a> Stem Cells Dev. 15 (5), 631-639 (2006).</li><li>Anderson, D., 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=Transgenic+enrichment+of+cardiomyocytes+from+human+embryonic+stem+cells.">Transgenic enrichment of cardiomyocytes from human embryonic stem cells.</a> Mol Ther. 15 (11), 2027-2036 (2007).</li><li>Huber, I., 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=Identification+and+selection+of+cardiomyocytes+during+human+embryonic+stem+cell+differentiation.">Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.</a> FASEB J. 21 (10), 2551-2563 (2007).</li><li>Kita-Matsuo, H., 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=Lentiviral+vectors+and+protocols+for+creation+of+stable+hESC+lines+for+fluorescent+tracking+and+drug+resistance+selection+of+cardiomyocytes.">Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.</a> PLoS One. 4 (4), e5046 (2009).</li><li>Choi, K. D., 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=Hematopoietic+and+endothelial+differentiation+of+human+induced+pluripotent+stem+cells.">Hematopoietic and endothelial differentiation of human induced pluripotent stem cells.</a> Stem Cells. 27 (3), 559-567 (2009).</li><li>Woll, P. 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=Wnt+signaling+promotes+hematoendothelial+cell+development+from+human+embryonic+stem+cells.">Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells.</a> Blood. 111 (1), 122-131 (2008).</li><li>Li, Z., Hu, S., Ghosh, Z., Han, Z., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+and+expression+profiling+of+human+induced+pluripotent+stem+cell-+and+embryonic+stem+cell-derived+endothelial+cells.">Functional characterization and expression profiling of human induced pluripotent stem cell- and embryonic stem cell-derived endothelial cells.</a> Stem Cells Dev. 20 (10), 1701-1710 (2011).</li><li>Rufaihah, A. 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=Endothelial+cells+derived+from+human+iPSCS+increase+capillary+density+and+improve+perfusion+in+a+mouse+model+of+peripheral+arterial+disease.">Endothelial cells derived from human iPSCS increase capillary density and improve perfusion in a mouse model of peripheral arterial disease.</a> Arterioscler Thromb Vasc Biol. 31 (11), e72-e79 (2011).</li><li>Beauchamp, J. R., Morgan, J. E., Pagel, C. N., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+myoblast+transplantation+reveal+a+discrete+minority+of+precursors+with+stem+cell-like+properties+as+the+myogenic+source.">Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.</a> J Cell Biol. 144 (6), 1113-1122 (1999).</li><li>Qu, Z., 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=Development+of+approaches+to+improve+cell+survival+in+myoblast+transfer+therapy.">Development of approaches to improve cell survival in myoblast transfer therapy.</a> J Cell Biol. 142 (5), 1257-1267 (1998).</li><li>Tang, X. 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=Intracoronary+administration+of+cardiac+progenitor+cells+alleviates+left+ventricular+dysfunction+in+rats+with+a+30-day-old+infarction.">Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction.</a> Circulation. 121 (2), 293-305 (2010).</li><li>Zeng, 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=Bioenergetic+and+functional+consequences+of+bone+marrow-derived+multipotent+progenitor+cell+transplantation+in+hearts+with+postinfarction+left+ventricular+remodeling.">Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling.</a> Circulation. 115 (14), 1866-1875 (2007).</li><li>Davis, M. E., 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=Local+myocardial+insulin-like+growth+factor+1+(IGF-1)+delivery+with+biotinylated+peptide+nanofibers+improves+cell+therapy+for+myocardial+infarction.">Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction.</a> Proc Natl Acad Sci U S A. 103 (21), 8155-8160 (2006).</li><li>Li, Q., 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=Overexpression+of+insulin-like+growth+factor-1+in+mice+protects+from+myocyte+death+after+infarction,+attenuating+ventricular+dilation,+wall+stress,+and+cardiac+hypertrophy.">Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy.</a> J Clin Invest. 100 (8), 1991-1999 (1997).</li><li>Wang, L., Ma, W., Markovich, R., Chen, J. W., Wang, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cardiomyocyte+apoptotic+signaling+by+insulin-like+growth+factor+I.">Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I.</a> Circ Res. 83 (5), 516-522 (1998).</li><li>Chong, J. 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=Human+embryonic-stem-cell-derived+cardiomyocytes+regenerate+non-human+primate+hearts.">Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.</a> Nature. 510 (7504), 273-277 (2014).</li></ol>

Amélioration de rendement / pureté de hiPSC-CMs protocoles classiques pour la différenciation des cellules souches humaines dans CMs sont souvent limitées par un faible rendement et la pureté; par exemple, juste 35-66% des CSEh-CMs obtenu par Percoll séparation et la formation du corps cardiaque exprimé myosine lente chaîne lourde ou cTnT 6. La pureté des populations hiPSC-CM différenciées peut être sensiblement augmentée en sélectionnant pour l&#39;express...

Les cellules souches pluripotentes humaines induites (les hiPSCs) sont parmi les agents les plus prometteurs pour la thérapie cellulaire régénérative, car ils peuvent être différenciées en une gamme potentiellement illimitée et la quantité de cellules qui ne sont pas rejetées par le système immunitaire du patient. Cependant, leur capacité d&#39;auto-réplication et la différenciation peut aussi conduire à la formation de tumeurs et, par conséquent, hiPSCs doivent être pleinement différenciées en types de cellules spécifiques, tels que les cardiomyocytes (SGC), les cellules endothéliales (EC), et les cellules musculaires lisses (CML ), avant l&#39;administration. Une des méthodes les plus simples et les plus courantes de l&#39;administration des cellules est l&#39;injection intra-myocardique directe, mais le nombre de cellules transplantées qui sont greffées par le tissu myocardique natif est exceptionnellement bas. Une grande partie de cette attrition peut être attribuée à l&#39;environnement cytotoxique du tissu ischémique; Cependant, lorsque les cellules souches embryonnaires murines (CES) ont été injectés directement dans le myocarde des cœurs indemnes, oeul ~ 40% des 5 millions de cellules livrées ont été retenus pendant 3-5 h 1, ce qui suggère qu&#39;une proportion importante des cellules administrées a quitté le site d&#39;administration, peut - être parce qu&#39;ils ont été évincés par la piste de l&#39; aiguille par les hautes pressions produites au cours de la contraction du myocarde. Ici, nous présentons des méthodes nouvelles et sensiblement plus efficaces pour générer cardiomyocytes hiPSC dérivés (hiPSC-SGC) 2, les cellules endothéliales (hiPSC-CEs) 3, et les cellules musculaires lisses (CML) 4. Notamment, ce protocole hiPSC-SMC est le premier à imiter le large éventail de caractéristiques morphologiques et fonctionnelles observées dans somatique CML 5 en dirigeant les cellules vers un phénotype SMC essentiellement synthétique ou contractile. Nous fournissons également une méthode de livraison de cellules qui améliore le taux de cellules injectées de greffe en créant un contenant cytokine-fibrine pATCH sur le site d&#39;injection. Le patch semble améliorer à la fois la rétention des cellules, en obturant la voie de l&#39;aiguille pour empêcher les cellules de sortir du myocarde, et la survie des cellules, en libérant le facteur de croissance analogue à l&#39;insuline (IGF), pendant une période d&#39;au moins trois jours.

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Protocol 1</td>
			<td style="width:180px;"></td>
			<td style="width:155px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 medium</td>
			<td>Stem cell technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth-factor-reduced matrigel</td>
			<td>Corning lifescience</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Stem cell technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement, serum free</td>
			<td>Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BMP-4</td>
			<td>R&#38;D</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bFGF</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase IV</td>
			<td>Fisher Scientific</td>
			<td>NC0217889</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:163px;">Hanks Balanced Salt Solution (Dextrose, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 anhydrous)</td>
			<td>Fisher Scientific</td>
			<td>14175079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10438018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate</td>
			<td>Corning Lifescience</td>
			<td>356721</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm dish</td>
			<td>Corning Lifescience</td>
			<td>354732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene</td>
			<td>Fisher Scientific</td>
			<td>15040066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibrinogen</td>
			<td>Sigma-Aldrich</td>
			<td>F8630-5g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T7009-1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMB2 medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VEGF</td>
			<td>ProSpec-Tany</td>
			<td>CYT-241</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EPO</td>
			<td>Life Technologies</td>
			<td>PHC9431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TGF-&#223;</td>
			<td>Peprotech</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGM2-MV medium</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB-431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD31</td>
			<td>BD Bioscience</td>
			<td>BDB555445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD144</td>
			<td>BD Bioscience</td>
			<td>560411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>12565269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorff Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR99021</td>
			<td>Stem cell technologies</td>
			<td>720542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDGF-&#223;</td>
			<td>Prospec</td>
			<td>CYT-501-10ug</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olive oil</td>
			<td>Sigma-Aldrich</td>
			<td>O1514</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGF</td>
			<td>R&#38;D</td>
			<td>291-G1-01M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>15561020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heating plate</td>
			<td>Fisher Scientific</td>
			<td>SP88850200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"><img alt="ezh" src="/files/ftp_upload/55142/ezh.jpg" />-aminocaproic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A0420000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM medium</td>
			<td>Fisher Scientific</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>Fisher Scientific</td>
			<td>1482748</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Datex-Ohmeda</td>
			<td>47810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Ohio Medical</td>
			<td>V5A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defibrillator</td>
			<td>Physiol Control</td>
			<td>LIFEPAK 15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5T MRI</td>
			<td>General Electric</td>
			<td>Signa Horizon LX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7T MRI</td>
			<td>Siemens</td>
			<td>10018532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gadolinium Contrast Medium (Magnevist)</td>
			<td>Berlex</td>
			<td>50419-188-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-0 silk suture</td>
			<td>Ethilon</td>
			<td>685H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 silk suture</td>
			<td>Ethilon</td>
			<td>622H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 monofilament suture</td>
			<td>Ethilon</td>
			<td>627H</td>
			<td></td>
		</tr>
	</tbody>
</table>

pluripotent stem cell derived cardiac cells for myocardial repair

Humaines induite par les cellules souches pluripotentes (hiPSCs) doivent être entièrement différenciées en types de cellules spécifiques avant l&#39;administration, mais des protocoles classiques pour différencier hiPSCs en cardiomyocytes (hiPSC-SGC), les cellules endothéliales (hiPSC-CEs), et les cellules musculaires lisses (CML) sont souvent limitée par un faible rendement, la pureté, et / ou une mauvaise stabilité phénotypique. Ici, nous présentons de nouveaux protocoles pour générer hiPSC-CMs, -ECs et -SMCs qui sont sensiblement plus efficaces que les méthodes conventionnelles, ainsi qu&#39;une méthode pour combiner l&#39;injection de cellules avec un patch contenant une cytokine créée sur le site de l&#39;administration. Le patch améliore à la fois la rétention des cellules injectées, en obturant la voie de l&#39;aiguille pour empêcher les cellules d&#39;être pressé hors du myocarde, et la survie des cellules, en libérant le facteur de croissance analogue à l&#39;insuline (IGF), pendant une période prolongée. Dans un modèle porcin d&#39;infarctus lésions d&#39;ischémie-reperfusion, le taux de prise de greffe était plus de deux fois plus élevée lorsque lales cellules ont été administrés avec le patch contenant les cytokines sont comparés aux cellules sans correction, et un traitement avec à la fois les cellules et le patch, mais non avec les cellules seules, a été associée à une amélioration significative de la fonction cardiaque et la taille de l&#39;infarctus.

Caractérisation des Differentiated hiPSC-CMs, -ECs et -SMCs La capacité différentielle de hiPSCs ont été évaluées 2, 3, 4. Débit analyse cytométrie de la troponine T cardiaque (cTnT) expression suggèrent que la pureté de la finale population hiPSC-CM peut dépasser 90% (figure 1A, 1B, panneau ...

Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Nous présentons trois protocoles nouveaux et plus efficaces pour différencier les cellules souches pluripotentes humaines induites dans les cardiomyocytes, les cellules endothéliales et les cellules musculaires lisses et une méthode de distribution qui permet d&#39;améliorer la prise de greffe de cellules transplantées en combinant l&#39;injection de cellules avec la livraison de cytokines pièce à médiation.

Les cellules souches pluripotentes dérivées de cellules cardiaques pour réparation myocardique

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

The overall goal of this video is to introduce a technique for differentiating human pluripotent stem cells into cardiomyocytes, and to demonstrate the use of these cells for studying myocardial repair after an ischemia reperfusion injury. This method can help answer key questions about engineering heart tissue with human iPS cell derived from cardiac cells for myocardial repair. The main advantage of this procedure is that it allows scientists to collect purified cardiomyocytes, smooth muscle cells, and endothelial cells, and generate heart tissue with these progenitor cells.Ling Gao is a post-doc in Doctor Zhang's own lab in UAB and Greg Walcott is a cardiologist in UAB hospital. Trina is a MRI specialist in UAB hospital. To induce pluripotent stem cells into cardiomyocytes, first, coat the wells of a six-well plate with growth-factor reduced gelatinous protein mixture cooled to 4 degrees Celsius.Then, refrigerate the plate overnight at 4 degrees Celsius. The next day, aspirate the gelatinous protein mixture and seed the plates with 100 million stem cells per well. Never keep the stem cells out of the incubator for more than 15 minutes.Also, always use fresh media. In this case, use mTeSR1 medium, supplemented with 10 micromolar of ROCK inhibitor. Then, culture the cells with 5%carbon dioxide at 37 degrees Celsius, and refresh the medium daily by gently aspirating away the old medium without touching the cells, and using a transfer pipette to apply fresh medium.Grow the cells until they are 90%confluent. Three days into the differentiation process, start checking for clusters of contracting cells. As soon as contractions are noted, which can take a week or so, collect the cells.Count the contracting cells using a hemocytometer, and then culture the cells on 10 centimeter plates at a concentration of one million cells per milliliter. It is also possible to make endothelial cells from stem cells, as well as smooth muscle cells. The processes are different, but are carefully detailed in the text protocol.First, surgically induce a myocardial infarction in a swine heart. Briefly perform a fourth intercostal thoracotomy to expose the left anterior descending coronary artery. Then, occlude the coronary artery using a ligature for 60 minutes.Defibrillate the heart as necessary. Suspend five milligrams of microspheres in one milliliter of fibrinogen solution, and load this into a syringe. Third, fill another syringe with one milliliter of thrombin solution.After an hour has passed, remove the ligature used to create the myocardial infarction. Then, position a plastic ring on the epicardium of the infarcted region, and simultaneously inject the microspheres and fibrinogen and the thrombin solutions into the ring. Allow the mixture of solutions to solidify, which takes about 30 seconds.This makes the patch. Then, insert the needle of the cell-containing syringe through the patch into the infarcted myocardium and inject the cells. Now, close the muscle layers, subcutaneous tissue, and the chest wall with 30 or 20 Monocryl suture, depending on the animal's size.One week and four weeks after the procedure, evaluate the animal's cardiac function using a cardiac MRI. As described by the first presented protocol, human pluripotent stem cells were differentiated into cardiomyocytes. To assess their purity, flow cytometry for cardiac troponin T showed it was present in excess of 90%of cells.Other markers were also observed. Nearly all of the cells expressed slow myocin heavy chain, and nearly all the cells expressed alpha sarcomeric actin. About one quarter of the cells expressed 2v isoform of myosin light chain, a marker for ventricular cardiomyocytes.In comparing stem cell lines, it was found that the efficiency of the differentiation was substantially higher with cells from the GRiPS line than from the PCBC16iPS line. Using the patch-mediated cell transplantation protocol, a mixture of cells, made up of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells was injected into the injured tissue. Four weeks later, more cells were retained with the patch than without it.Within one week, treatment using the cell injection with the patch resulted in the most improvement of the infarct size. Also, there was no appreciable difference in injecting with just cardiomyocytes compared to injecting with three cell types. After watching this video, you should have a good understanding on how to differentiate human iPS cells into three different cardiac ligature cells, including cardiomyocytes, smooth muscle cells, and endothelial cells.You should also have a good understanding of how to generate human heart tissue with the progenitor cells for myocardial repair. Since the development, this technique has advanced investigations on myocardial repair and stem cell tissue engineering. It should be noted that when attempting this procedure, it is very important to use high quantity of iPS cells and their cardiac derivatives.

Research

JoVE Journal

Developmental Biology

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Génération de stimulateur cardiaque murin agrégats de cellules fondées sur la ES-Cell-programmation en combinaison avec MYH6-promoteur-sélection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

Recherche

Biologie du développement

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Génération de l&#39;intégration sans anthropiques cellules souches pluripotentes utilisant des kératinocytes cheveux dérivés

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Dérivation libre Feeder-de mélanocytes de pluripotentes humaines Cellules souches

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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

Production à grande échelle de cardiomyocytes à partir pluripotentes humaines cellules souches en utilisant un petit protocole Différenciation Molecule-Based hautement reproductible

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

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Différenciation des chondrocytes provenant des cellules souches pluripotentes induites par le sang périphérique dérivé du sang

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Formation de granulés chondrogéniques à partir de cellules souches pluripotentes induites par sang de cordon

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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

Rapide, réalisée la différenciation des cellules épithéliales pigmentaire rétinien de cellules souches pluripotentes humaines embryonnaires ou induit

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

Single-cell Photoconversion in Living Intact Zebrafish

Photoconversion cellule unique chez le poisson zèbre Intact de la vie

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

À l’aide de l’homme induit des hépatocytes-comme dérivé de cellules souches pluripotentes for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Un modèle de souris chimériques du foie humain de l’hypercholestérolémie utilisant des hépatocytes de dérivés de cellules souches pluripotentes induites

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...