Name: enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity
Uploaded: 2019-07-10T12:30:00
Description: Watch this Scientific Journal Video about Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity at JoVE.com

Forfatterne takker Dr. Joseph C. Wu (Stanford University) for venligt at give Fluc-GFP konstruere og Dr. Yanwen Liu for fremragende teknisk assistance. Denne undersøgelse er støttet af National Institutes of Health RO1 tilskud HL95077, HL114120, HL131017, HL138023, UO1 HL134764 (til J.Z.), og HL121206A1 (til L.Z.), og en R56 Grant HL142627 (til W.Z.), en amerikansk hjerte forening videnskabsmand udvikling Grant 16SDG30410018, og University of Alabama på Birmingham Faculty Development Grant (til W.Z.).

Alle dyreforsøg i denne undersøgelse blev godkendt af den institutionelle dyrepleje-og brugs Komité (IACUC) fra University of Alabama i Birmingham og var baseret på de nationale institutter for sundheds laboratoriets dyrepleje og retningslinjer for anvendelse (NIH publication No 85-23).
1. forberedelse af kultur medier og kultur plader
<ol><li>
Medium forberedelse
	<ol>
<li>For hiPSC medium blandes 400 mL humant pluripotente stamcelle (hPSC) basal medium (tabel o...

<ol>
	<li>Menasche, P., 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=Towards+a+clinical+use+of+human+embryonic+stem+cell-derived+cardiac+progenitors:+a+translational+experience.">Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience.</a> European Heart Journal. 36 (12), 743-750 (2015).</li><li>Burridge, P. W., Keller, G., Gold, J. D., 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=Production+of+de+novo+cardiomyocytes:+human+pluripotent+stem+cell+differentiation+and+direct+reprogramming.">Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming.</a> Cell Stem Cell. 10 (1), 16-28 (2012).</li><li>Frisch, S. M., Francis, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+epithelial+cell-matrix+interactions+induces+apoptosis.">Disruption of epithelial cell-matrix interactions induces apoptosis.</a> Journal of Cell Biology. 124 (4), 619-626 (1994).</li><li>Haun, F., 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+of+a+novel+anoikis+signalling+pathway+using+the+fungal+virulence+factor+gliotoxin.">Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin.</a> Nature Communications. 9 (1), 3524 (2018).</li><li>Ohashi, 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=Rho-associated+kinase+ROCK+activates+LIM-kinase+1+by+phosphorylation+at+threonine+508+within+the+activation+loop.">Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop.</a> Journal of Biological Chemistry. 275 (5), 3577-3582 (2000).</li><li>Katoh, K., Kano, Y., Noda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-associated+kinase-dependent+contraction+of+stress+fibres+and+the+organization+of+focal+adhesions.">Rho-associated kinase-dependent contraction of stress fibres and the organization of focal adhesions.</a> Journal of The Royal Society Interface. 8 (56), 305-311 (2011).</li><li>Paoli, P., Giannoni, E., Chiarugi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoikis+molecular+pathways+and+its+role+in+cancer+progression.">Anoikis molecular pathways and its role in cancer progression.</a> Biochimica et Biophysica Acta. 1833 (12), 3481-3498 (2013).</li><li>Legate, K. R., Fassler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+that+regulate+adaptor+binding+to+beta-integrin+cytoplasmic+tails.">Mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails.</a> Journal of Cell Science. 122 (Pt 2), 187-198 (2009).</li><li>Watanabe, 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+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</li><li>Emre, 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=The+ROCK+inhibitor+Y-27632+improves+recovery+of+human+embryonic+stem+cells+after+fluorescence-activated+cell+sorting+with+multiple+cell+surface+markers.">The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.</a> PLoS One. 5 (8), e12148 (2010).</li><li>Ni, Y., Qin, Y., Fang, Z., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+Inhibitor+Y-27632+Promotes+Human+Retinal+Pigment+Epithelium+Survival+by+Altering+Cellular+Biomechanical+Properties.">ROCK Inhibitor Y-27632 Promotes Human Retinal Pigment Epithelium Survival by Altering Cellular Biomechanical Properties.</a> Current Molecular Medicine. 17 (9), 637-646 (2017).</li><li>Laflamme, M. 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=Cardiomyocytes+derived+from+human+embryonic+stem+cells+in+pro-survival+factors+enhance+function+of+infarcted+rat+hearts.">Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.</a> Nature Biotechnology. 25 (9), 1015-1024 (2007).</li><li>Zhu, W., Zhao, M., Mattapally, S., Chen, S., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCND2+Overexpression+Enhances+the+Regenerative+Potency+of+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes:+Remuscularization+of+Injured+Ventricle.">CCND2 Overexpression Enhances the Regenerative Potency of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Remuscularization of Injured Ventricle.</a> Circulation Research. 122 (1), 88-96 (2018).</li><li>Song, 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=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485 (7400), 599-604 (2012).</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>Tohyama, 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=Glutamine+Oxidation+Is+Indispensable+for+Survival+of+Human+Pluripotent+Stem+Cells.">Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells.</a> Cell Metabolism. 23 (4), 663-674 (2016).</li><li>Ong, S. G., 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=Microfluidic+Single-Cell+Analysis+of+Transplanted+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes+After+Acute+Myocardial+Infarction.">Microfluidic Single-Cell Analysis of Transplanted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes After Acute Myocardial Infarction.</a> Circulation. 132 (8), 762-771 (2015).</li><li>Zhao, M., 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=Y-27632+Preconditioning+Enhances+Transplantation+of+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes+in+Myocardial+Infarction+Mice.">Y-27632 Preconditioning Enhances Transplantation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Myocardial Infarction Mice.</a> Cardiovascular Research. , (2018).</li><li>Tohyama, 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=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell Stem Cell. 12 (1), 127-137 (2013).</li><li>Silginer, M., Weller, M., Ziegler, U., Roth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+inhibition+promotes+atypical+anoikis+in+glioma+cells.">Integrin inhibition promotes atypical anoikis in glioma cells.</a> Cell Death &amp; Disease. 5, e1012 (2014).</li><li>Lelievre, E. C., 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=N-cadherin+mediates+neuronal+cell+survival+through+Bim+down-regulation.">N-cadherin mediates neuronal cell survival through Bim down-regulation.</a> PLoS One. 7 (3), e33206 (2012).</li><li>Murata, 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=Increase+in+cell+motility+by+carbon+ion+irradiation+via+the+Rho+signaling+pathway+and+its+inhibition+by+the+ROCK+inhibitor+Y-27632+in+lung+adenocarcinoma+A549+cells.">Increase in cell motility by carbon ion irradiation via the Rho signaling pathway and its inhibition by the ROCK inhibitor Y-27632 in lung adenocarcinoma A549 cells.</a> Journal of Radiation Research. 55 (4), 658-664 (2014).</li><li>Srivastava, K., Shao, B., Bayraktutan, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC-beta+exacerbates+in+vitro+brain+barrier+damage+in+hyperglycemic+settings+via+regulation+of+RhoA/Rho-kinase/MLC2+pathway.">PKC-beta exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 33 (12), 1928-1936 (2013).</li></ol>

De vigtigste trin i denne undersøgelse omfatter at opnå ren hiPSC-CMs, forbedre aktiviteten af hiPSC-CMs gennem Y-27632 forbehandling, og endelig, transplantere en præcis mængde hiPSC-CMs i en mus MI model.
De centrale spørgsmål, der blev behandlet her, var, at vi først optimerede de glucosefri rensningsmetoder19 og etablerede et nyt effektivt rensningssystem. System proceduren omfattede anvendelse af celle dissociations enzymer, genbeplantning af celler i gelati...

Stamcelle baserede terapier har vist et betydeligt potentiale som en behandling for hjerte skader forårsaget af MI1. Brugen af differentierede hiPSCs giver en uudtømmelig kilde til hiPSC-CMs2 og åbner døren for en hurtig udvikling af banebrydende behandlinger. Men, mange begrænsninger for terapeutisk oversættelse forbliver, herunder udfordringen med den alvorligt lave engraftment sats af implanterede celler.
Dissocierende celler med trypsin initierer anoikis3, som kun accelereres, når disse celler injiceres i barske miljøer som den iskæmiske myokardiet, hvor det hypoxiske miljø accelererer kursen mod celledød. Af de resterende celler, en stor del vaskes ud fra implantationsstedet ind i blodbanen og spredes i hele periferien. En af de vigtigste apoptotiske veje er RhoA/ROCK pathway4. Baseret på tidligere forskning, rhoa/rock pathway regulerer actin cytoskelet organisation5,6, som er ansvarlig for celle dysfunktion7,8. Rock inhibitor Y-27632 er meget udbredt under somatisk og stamcelle dissociation og passaging, at øge celle vedhæftning og reducere celle apoptose9,10,11. I dette studie anvendes Y-27632 til behandling af hiPSC-CMs før transplantation i et forsøg på at øge cellernes engraftment.
Der er fastlagt flere metoder til forbedring af celle engraftment, såsom varmechok og kælder membran matrix belægning12. Bortset fra disse metoder kan genteknologi også fremme kardiomyocyt proliferation13 eller vende nonmyokardial celler til kardiomyocytter14. Fra Bioengineering perspektiv, cardiomyocytter seedet på en biomaterialet stilladset for at forbedre transplantationen effektivitet15. Desværre er de fleste af disse metoder komplicerede og dyre. Tværtimod er den metode, der foreslås her, enkel, omkostningseffektiv og effektiv, og den kan bruges som basal behandling før transplantation, samt i konjugering med andre teknologier.

<table>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase (stem cell detachment solution)</td>
			<td>STEMCELL Technologies</td>
			<td>#07920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 minus insulin</td>
			<td>Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Fisher Scientific</td>
			<td>17-504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stem Cell Technologies</td>
			<td>72054</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1x), high glucose, HEPES, no phenol red</td>
			<td>Thermofisher</td>
			<td>20163029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM (calcium indicator)</td>
			<td>Invitrogen/Thermofisher</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-free RPMI 1640</td>
			<td>Fisher Scientific</td>
			<td>11879020</td>
			<td></td>
		</tr>
		<tr>
			<td>IWR1</td>
			<td>Stem Cell Technologies</td>
			<td>72562</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (extracellular matrix )</td>
			<td>Fisher Scientific</td>
			<td>CB-40230C</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR (human pluripotent stem cells medium)</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-strep antibiotic</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 (surfactant polyol)</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Rho activator II</td>
			<td>Cytoskeleton</td>
			<td>CN03</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium DL-lactate</td>
			<td>Sigma-Aldrich</td>
			<td>L4263</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE (cell-dissociation enzymes)</td>
			<td>Fisher Scientific</td>
			<td>12-605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment and Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina III Bioluminescence Instruments</td>
			<td>PerkinElmer</td>
			<td>CLS136334</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm Coverslips</td>
			<td>Warner</td>
			<td>CS-15R15</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo Scientific</td>
			<td>NX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Automatic Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply</td>
			<td>BIO-RAD</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresence Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>pco</td>
			<td>1200 s</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scan Head</td>
			<td>Olympus</td>
			<td>FV-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Profile Open Bath Chamber (mounts into above microincubation system)</td>
			<td>Warner Instruments</td>
			<td>RC-42LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Microincubation System</td>
			<td>Warner Instruments</td>
			<td>DH-40iL</td>
			<td></td>
		</tr>
		<tr>
			<td>Minivent Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>845</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD/SCID mice</td>
			<td>Jackson Laboratory</td>
			<td>001303</td>
			<td></td>
		</tr>
		<tr>
			<td>Precast Protein Gels</td>
			<td>BIO-RAD</td>
			<td>4561033</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Transfer Packs</td>
			<td>BIO-RAD</td>
			<td>1704156</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot System</td>
			<td>BIO-RAD</td>
			<td>Trans-Blot Turbo</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human Nucleolin (Alexa Fluor 647)</td>
			<td>Abcam</td>
			<td>ab198580</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin T</td>
			<td>R&#38;D Systems</td>
			<td>MAB1874</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin C</td>
			<td>Abcam</td>
			<td>ab137130</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin I</td>
			<td>Abcam</td>
			<td>ab47003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5-donkey anti-mouse</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>715-175-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-donkey anti-rabbit</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>711-165-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Fitc-donkey anti-mouse</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>715-095-150</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>Abcam</td>
			<td>ab22555</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cardiac Troponin T</td>
			<td>Abcam</td>
			<td>ab91605</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrin &#946;1</td>
			<td>Abcam</td>
			<td>ab24693</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>EMD Millipore</td>
			<td>ab9260</td>
			<td></td>
		</tr>
		<tr>
			<td>N-cadherin</td>
			<td>Abcam</td>
			<td>ab18203</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Myosin Light Chain 2</td>
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enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity

En afgørende faktor for at forbedre cellulære terapi effektivitet for Myokardie regenerering er at sikkert og effektivt øge cellen engraftment sats. Y-27632 er en meget potent hæmmer af Rho-associeret, coiled-spole-holdige protein kinase (RhoA/ROCK) og bruges til at forhindre dissociation-induceret celle apoptose (anoikis). Vi viser, at Y-27632 forbehandling for menneskeskabte pluripotente stamcelle-afledte kardiomyocytter (hiPSC-CMs+ Ri) forud for implantation resulterer i en celle engraftment rate forbedring i en musemodel af akut MYOKARDIEINFARKT (MI). Her beskriver vi en komplet procedure af hiPSC-CMs differentiering, rensning, og celle forbehandling med Y-27632, samt den resulterende celle sammentrækning, calcium forbigående målinger, og transplantation i mus MI modeller. Den foreslåede metode giver en enkel, sikker, effektiv og billig metode, som i væsentlig grad øger cellengraftment sats. Denne metode kan ikke kun anvendes i forbindelse med andre metoder til yderligere at forbedre celletransplantation effektivitet, men også giver et gunstigt grundlag for studiet af mekanismerne i andre hjertesygdomme.

Det hipsc-CMS, der anvendes i dette studie, stammer fra menneskelig oprindelse med der reporter-genet; Overlevelsesraten for de transplanterede celler in vivo blev derfor påvist ved bioluminescens Imaging (BLI)17 (figur 1a, B). For histologiske hjerte sektioner blev Human-specifikke kardiale troponin T (hcTnT) og humane nukleare antigen (HNA) dobbelt positive celler klassificeret som enpodet hiPSC-CMs (<strong class="...

Watch this Scientific Journal Video about Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity at JoVE.com

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

I denne protokol viser og uddyber vi, hvordan man bruger menneskeskabte pluripotente stamceller til differentiering og rensning af kardiomyocytter, og yderligere om, hvordan man kan forbedre sin transplantations effektivitet med Rho-associeret proteinkinasehæmmer forbehandling i en mus myokardieinfarkt model.

Forbedring af engraftment af humane inducerede pluripotente stamcelle afledte Kardiomyocytter via en forbigående hæmning af Rho Kinaseaktivitet

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Vores data viser, at forbigående hæmning af Rho-kinase aktivitet øger vedhæftning, overlevelse, og engraftment af hiPSC-afledte kardiomyocytter efter at være blevet transplanteret ind i infarkted dyr hjerter. Vi leverer en enkel, billig, mens højeffektiv metode til at øge engraftment af transplanterede hiPSC-afledte kardiomyocytter i de skadede dyrehjerter. Da denne teknik kan forbedre celle engraftment satser sikkert og effektivt i akut hjerteinfarkt og hjertesvigt, det bidrager til de deraf følgende forbedringer i hjertefunktion, vaskulariseringer, og apoptose.Selv om vi kun testede vores protokoller i hiPSCs, kan disse metoder anvendes på andre typer af celler, såsom embryonale stamceller, mesenkymale stamceller osv. Dosis og varighed af behandling med Y-27632 er kritisk. Den visuelle demonstration kan demonstrere i detaljer alle vigtige trin i protokollen, hvilket er til gavn for en person, der aldrig har udført denne teknik før.På dag 21 efter hiPSC-CM differentiering, aspirere mediet og vask cellerne med steril PBS én gang. Inkuber cellerne med 0,5 milliliter celleafisationenzymer pr. brønd ved 37 grader Celsius i fem til syv minutter. Efter inkubationstiden pipetteres celleaffjedringen gentagne gange med en pipette med en milliliter for grundigt at adskille cellerne.Derefter tilsættes en milliliter neutraliserende opløsning til hver brønd, indsamle celleblandingen i et 15-milliliter centrifugerør, og centrifuge ved 200 gange g i tre minutter. Efter centrifugering, kassér supernatant og resuspend cellerne i neutraliserende opløsning. Derefter genplantes cellerne på gelatinebelagte, seks-brønds plader med en tæthed på to millioner celler pr. brønd.Efter 24 til 48 timer udskiftes kulturmediet med rensemediet i tre til fem dage. Før transplantation, kultur cellerne i behandlingsgruppen i 12 timer i RB-plus medium suppleret med 10-mikromolar Y-27632. På samme måde udføres verapamilbehandling på cellerne i RB-plus-mediet med en mikromol af verapamil i 12 timer.Efter behandlingen vaskes hiPSC-CMs med PBS én gang, og cellerne inkuberes med 0,5 mikroliter af celleafkost enzymer pr. brønd i højst to minutter. Gentagne gange pipette celle suspensionen ved hjælp af en en-milliliter pipette til grundigt at adskille cellerne. Dernæst neutralisere cellerne med neutraliserende opløsning, indsamle celleblandingen i en 15-milliliter centrifuge rør, og centrifuge ved 200 gange g i tre minutter.Efter centrifugering opsplitning af cellerne i PBS i en koncentration på 0,1 millioner celler pr. fem mikroliter som forberedelse til injektion. Umiddelbart efter myokardieinuktion skal der injiceres fem mikroliter af kontrol hiPSC-CMs, Y-27632-forbehandlet hiPSC-CMs, verapamil-behandlede hiPSC-CMs eller en tilsvarende mængde PBS i musens myokardiet på hvert sted, en i det infarkte område og to i områderne omkring infarct. 12 timer før du udfører calcium forbigående og kontraktilitet detektion, tilføjes 10-mikromolar Y-27632, 100-nanomolar RA, en-mikromolar verapamil, eller en tilsvarende mængde PBS i kulturmediet af hiPSC-CMs.Dernæst kasseres mediet, og vask pladen med PBS én gang. Skift mediet til en phenol rødfri DMEM, der indeholder 0,02 vægt pr. volumenprocent af overfladeaktiv polyol, fem mikromolarcalciumindikator, og den tilsvarende Y-27632, RA, verapamil eller et tilsvarende volumen PBS, og inkuber pladen ved 37 grader Celsius i 30 minutter. Efter inkubationen kasseres supernatanten, cellerne vaskes med det farvefri DMEM tre gange, og mediet hvile i 30 minutter for at afstælle kortlægningsfarvestoffet.Placer celle-seedede coverslip i et åbent bad kammer, og indsætte kammeret i en mikroincubation system ækvibreret til 37 grader Celsius med en automatisk temperaturregulator. Gennemgå det med Tyroders opløsning, og tilsæt løbende RI, RA eller PBS til perfusionsopløsningen. Brug et omvendt fluorescensmikroskop og et laserscanningshoved til at registrere spontan calciumtransient ved at anvende en x-t-linjescanning.Optag den spontane sammentrækning af hiPSC-CMs ved hjælp af den differenskontrastsammenligningskontraffunktionalitet i konfokale mikroskop. Overlevelsesraten for de transplanterede celler blev bekræftet af den øgede luciferaseaktivitet. Den øgede menneskelige hjertetroponin T og humane nukleare antigen udtryk viste, at Y-27632 forbehandling kunne forbedre celle engraftment sats.Y-27632 forbehandlede celler udviste en større og mere defineret stangformet cytoskeletal struktur. Også, Y-27632 forbehandling faldt TUNEL-positive celler, hvilket indikerer den reducerede transplanterede hiPSC-CM apoptose in vivo. Western blot foreslog, at Y-27632 forbehandling øget udtryk for integrin beta-1 og N-cadherin og faldt udtrykket af fosfor-myosin lyskæde 2.Denne ændring blev normaliseret 72 timer efter Y-27632 tilbagetrækning. In vitro mus HL-1 celle vedhæftet fil eksperimenter viste, at Y-27632 forbehandling betydeligt øget hiPSC-CM overholdelse i forhold til HL-1. Y-27632 forbehandling reducerede kontraktilekraften, den maksimale calciumtransient fluorescens og calciumtransientens varighed.Y-27632 forbehandling reguleret cardiomyocyte sammentrækning ved at reducere udtrykket af cTnI og cTnT. Svarende til Y-27632, verapamil forbehandling øget luciferase aktivitet og antallet af hcTnT/HNA dobbelt-positive celler. Det vigtigste skridt er at dyrke celler i behandlingsgruppen i 12 timer i Rb-plus medium suppleret med 10-mikromolar Y-27632.Baseret på denne metode, langsom frigivelse af Y-27632 at yderligere øge cellen engraftment sats er en lovende strategi. Denne teknik baner vejen for forskere at studere fysiologi og funktion af hiPSC af CMs in vivo. Det lille molekyle CHIR99021, der anvendes til kardiomyocytter differentiering, er farligt, hvilket kan forårsage irritation af luftvejene.Vær venlig ikke at indtage eller inhalere på nogen måde.

Research

JoVE Journal

Biochemistry

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

Kvantificering af Bakteriel Histidin kinase Autophosphorylering Ved hjælp af en Nitrocellulose-bindingsassay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Assaying Protein Kinase Activity with Radiolabeled ATP

Analyse af proteinkinaseaktivitet med radiomærket ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identifikation af Cyclin-afhængig Kinase 1 specifikke fosforylering websteder ved en In Vitro -Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Anaerob Protein oprensning og kinetisk analyse via ilt elektrode til at studere DesB Dioxygenase aktivitet og hæmning

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Human perifert blod Neutrofile Isolation for at forhøre Parkinsons Associated LRRK2 Kinase Pathway ved at vurdere Rab10 fosforylering

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

Identifikation af roman CK2 Kinase substrater ved hjælp af en alsidig biokemiske tilgang

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

At studere RNA Interactors af Protein Kinase RNA-aktiveret under pattedyr cellecyklus

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Enkelt trin berigelse af en TAP-Tagged Histon Deacetylase af Filamentous svamp Aspergillus nidulans for enzymatisk aktivitetsanalyse

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Karakterisering på molekyleniveau ved hjælp af robuste biokemiske tilgange af et nyt kinase protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Kinasehæmmer screening i selv monterede humane protein mikrosystemer

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...