Name: generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells
Uploaded: 2018-06-19T14:00:00
Description: Watch this Scientific Journal Video about Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells at JoVE.com

Vi tackar medlemmar av Colas lab för bra diskussioner och kritiska omdömen om manuskriptet. Denna studie stöddes av NIH/NIEHS R44ES023521-02 och CIRM disk2-10110 beviljar för Dr. Colas.

1. Id1 Virus förberedelse och infektion
<ol><li>Generera Id1 överuttryck lentivirus genom samarbete transfecting pCMVDR8.74, pMD2.G och pCDH-EF1-Id1-PGK-PuroR (Addgene: plasmid #107735) i HEK293T celler. Samla in viruspartiklar, filtrat, rena från supernatanten och förvaras vid-80 ° C som i Kitamura o.a. (2003) 19. Alternativt producera Id1 lentivirus kommersiellt genom att skicka pCDH-EF1-Id1-PGK-PuroR till en lentivirus-producerande leverantör. Varning: Följ lentiviral säke...

<ol>
	<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, 273-277 (2014).</li><li>Kodo, 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=iPSC-derived+cardiomyocytes+reveal+abnormal+TGF-beta+signalling+in+left+ventricular+non-compaction+cardiomyopathy.">iPSC-derived cardiomyocytes reveal abnormal TGF-beta signalling in left ventricular non-compaction cardiomyopathy.</a> Nature cell biology. 18, 1031-1042 (2016).</li><li>Kim, 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=Studying+arrhythmogenic+right+ventricular+dysplasia+with+patient-specific+iPSCs.">Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.</a> Nature. 494, 105-110 (2013).</li><li>Colas, A. 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=Whole-genome+microRNA+screening+identifies+let-7+and+mir-18+as+regulators+of+germ+layer+formation+during+early+embryogenesis.">Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis.</a> Genes &amp; development. 26, 2567-2579 (2012).</li><li>Cunningham, T. 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=Id+genes+are+essential+for+early+heart+formation.">Id genes are essential for early heart formation.</a> Genes &amp; development. , (2017).</li><li>McKeithan, W. L., Colas, A. R., Bushway, P. J., Ray, S., Mercola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+generation+of+multipotent+mesoderm+(Kdr+)+progenitor+cells+in+mouse+embryonic+stem+cells+for+functional+genomics+screening.">Serum-free generation of multipotent mesoderm (Kdr+) progenitor cells in mouse embryonic stem cells for functional genomics screening.</a> Current protocols in stem cell biology. , 13 (2012).</li><li>McKeithan, W. 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=An+automated+platform+for+assessment+of+congenital+and+drug-induced+arrhythmia+with+hipsc-derived+cardiomyocytes.">An automated platform for assessment of congenital and drug-induced arrhythmia with hipsc-derived cardiomyocytes.</a> Frontiers in physiology. 8, 766 (2017).</li><li>Sharma, 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=High-throughput+screening+of+tyrosine+kinase+inhibitor+cardiotoxicity+with+human+induced+pluripotent+stem+cells.">High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.</a> Science translational medicine. 9, (2017).</li><li>Burridge, P. W., 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=Chemically+defined+generation+of+human+cardiomyocytes.">Chemically defined generation of human cardiomyocytes.</a> Nature methods. 11, 855-860 (2014).</li><li>Kattman, S. 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=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell stem cell. 8, 228-240 (2011).</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, 1015-1024 (2007).</li><li>Lian, X., 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=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 1848-1857 (2012).</li><li>Willems, 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=Small+molecule-mediated+TGF-beta+type+II+receptor+degradation+promotes+cardiomyogenesis+in+embryonic+stem+cells.">Small molecule-mediated TGF-beta type II receptor degradation promotes cardiomyogenesis in embryonic stem cells.</a> Cell stem cell. 11, 242-252 (2012).</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=Human+cardiovascular+progenitor+cells+develop+from+a+KDR++embryonic-stem-cell-derived+population.">Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.</a> Nature. 453, 524-528 (2008).</li><li>Palpant, N. 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=Generating+high-purity+cardiac+and+endothelial+derivatives+from+patterned+mesoderm+using+human+pluripotent+stem+cells.">Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells.</a> Nature protocols. 12, 15-31 (2017).</li><li>DeLaughter, D. 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=Single-cell+resolution+of+temporal+gene+expression+during+heart+development.">Single-cell resolution of temporal gene expression during heart development.</a> Developmental cell. 39, 480-490 (2016).</li><li>Liu, X., 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+complex+genetics+of+hypoplastic+left+heart+syndrome.">The complex genetics of hypoplastic left heart syndrome.</a> Nature genetics. 49, 1152-1159 (2017).</li><li>Corrado, D., Link, M. S., Calkins, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrhythmogenic+right+ventricular+cardiomyopathy.">Arrhythmogenic right ventricular cardiomyopathy.</a> New England journal of medicine. 376, 1489-1490 (2017).</li><li>Kitamura, T., 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=Retrovirus-mediated+gene+transfer+and+expression+cloning:+powerful+tools+in+functional+genomics.">Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics.</a> Experimental hematology. 31, 1007-1014 (2003).</li></ol>

För framgångsrika differentieringar, se till att noga följa instruktionerna ovan. Dessutom här lyfter vi fram viktiga parametrar som starkt påverkar differentiering utfall. Innan du börjar en differentiering, följande tre morfologiska parametrar bör följas: en stam morfologi av hPSCsId1, en hög cellulära packning och en hög sammanflödet (> 90%) av kulturen på dag 0. I detta avseende optimal differentiering villkor skapas bäst av plätering dissocierade hPSCsId1 som enstaka celler och ...

Storskalig produktion av mänskliga pluripotenta stamceller (hPSCs)-härledda hjärt stamfäder och hjärtmuskelcellerna är en förutsättning för stamceller-baserade behandlingar1, sjukdom modellering2,3 och snabba karakterisering av nya vägar reglerar hjärt differentiering4,5,6 och fysiologi7,8. Även om ett antal studier9,10,11,12,13,14beskrivit,15 tidigare högeffektiva hjärt differentiering protokoll från hPSCs, har ingen tagit upp hjärtat fältet ursprung resulterande hjärtmuskelceller, trots identifiering av betydande molekylära skillnader mellan vänster (första hjärtat fält) och höger (andra hjärtat field) ventrikulär hjärtmuskelcellerna16 och förekomsten av hjärtat branschvisa medfödda hjärtsjukdomar; dvs, hypoplastisk vänster hjärta syndrom17 eller Högerkammarfunktion höger kammare dysplasi18. Således generationen av hjärt stamfäder och hjärtmuskelceller av definierade hjärtat fältet ursprung från hPSCs blir en nödvändighet för att öka deras relevans som terapeutiska och sjukdom modelleringsverktyg.
Protokollet bygger på den konstituerande överuttryck av Id1, en nyligen identifierade5 första hjärtat fält-ange cue som i kombination med Activin A, är både nödvändig och tillräcklig för att inleda cardiogenesis i hPSCs. Noterbart Cunningham et al. (2017) 5 Visa att Id1-inducerad föräldraparets särskilt uttrycka första hjärtat fältet (HCN4, TBX5) men inte andra hjärtat Fältmarkörer (SIX2, ISL1) som de genomgår hjärt differentiering. Dessutom visar författarna också att transgena möss embryon saknas hela Id familjen av gener (Id1-4), utveckla utan att bilda första hjärtat fältet hjärt progenitorceller, medan fler mediala och bakre hjärt stamfäder ( andra hjärtat fältet) kan fortfarande bilda, tyder därmed på att ID-proteiner är nödvändigt att inleda första hjärtat fältet cardiogenesis i vivo. Bekvämt, Id1-inducerad föräldraparets kan förvaras i fryst tillstånd och spontant differentieras till hjärtmuskelcellerna visar ventrikulära-liknande egenskaper, inklusive ventrikulär-specifika markörer (IRX4, MYL2) uttryck och ventrikulär-liknande handlingspänningar.
Här beskriver vi en enkel och skalbar metod för att generera första hjärtat fält-liknande (FHF-L) hjärt stamfäder och ventrikulära-liknande hjärtmuskelcellerna från Id1 överuttryck av hPSCs. Ett viktigt inslag i detta protokoll är möjligheten att avskilja hjärt stamceller generation från efterföljande hjärtmuskelcellen produktion med hjälp av ett praktiskt frysförvaring steg. Sammanfattningsvis, detta protokoll Detaljer nödvändiga åtgärder för att (1) generera Id1-överuttryck hPSCs, (2) generera FHF-L hjärt stamfäder från hPSCs, (3) frysa resulterande stamfäder och (4) återuppta FHF-L hjärt stamceller differentiering och generera höganrikat (> 70 – 90%) slog ventrikulära-liknande hjärtmuskelcellerna.

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			<td height="20" style="height:20px;width:204px;">ACTC1 antibody</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:119px;">A7811</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>Stem Cell Technologies</td>
			<td>Hu Recom Activin A</td>
			<td></td>
		</tr>
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			<td height="20" style="height:20px;">Antibiotic Antimycotic (Anti-Anti)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement w/o - insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement w/o - vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CDH5 antibody</td>
			<td style="width:144px;">R&#38;D Systems</td>
			<td>AF938</td>
			<td></td>
		</tr>
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			<td height="20" style="height:20px;">CryoStor CS10</td>
			<td>Stem Cell Technologies</td>
			<td>7930</td>
			<td style="width:444px;">Cryopreservation reagent</td>
		</tr>
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			<td height="20" style="height:20px;">DMEM high Glucose</td>
			<td>Mediatech</td>
			<td>10-013-CV&#160;</td>
			<td></td>
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			<td height="20" style="height:20px;">DPBS w/ Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
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			<td height="20" style="height:20px;">EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575-038</td>
			<td></td>
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			<td height="20" style="height:20px;">FBS</td>
			<td>VWR</td>
			<td>89510-186</td>
			<td></td>
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			<td height="20" style="height:20px;">FluoVolt membrane potential kit&#160;&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>F10488</td>
			<td>For optical action potential acquisition, please refer to McKeithan et al. 2017</td>
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		<tr height="20">
			<td height="20" style="height:20px;">KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel, Growth Factor Reduced</td>
			<td>Corning</td>
			<td>356231</td>
			<td style="width:444px;">Coating reagent</td>
		</tr>
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			<td height="20" style="height:20px;">mTeSR1 media kit</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS w/o Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
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			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puromycin&#160;</td>
			<td>Acros</td>
			<td>227422500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td style="width:444px;">Enzyme-free dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TAGLN antibody</td>
			<td style="width:144px;">Abcam</td>
			<td>ab14106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thiazovivin</td>
			<td>Stem Cell Technologies</td>
			<td>72254</td>
			<td style="width:444px;">RHO/ROCK pathway inhibitor</td>
		</tr>
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			<td height="20" style="height:20px;">TrypLE Express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605 -010</td>
			<td style="width:444px;">1X enzyme-containing dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyrodes solution mix packets&#160;&#160;</td>
			<td>Sigma</td>
			<td>T2145-10X1L</td>
			<td>(For optical action potential acquisition, please refer to McKeithan et al. 2017)</td>
		</tr>
	</tbody>
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generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells

Generering av stora mängder funktionella mänskliga pluripotenta stamceller-derived hjärt stamfäder och hjärtmuskelceller definierade hjärtat fältet ursprung är en förutsättning för cellbaserade hjärt terapier och sjukdom modellering. Vi har nyligen visat att Id gener är både nödvändigt och tillräckligt att ange första hjärtat fältet föräldraparets under ryggradsdjur utveckling. Denna differentiering-protokollet utnyttjar dessa fynd och använder Id1 överuttryck i kombination med Activin A som potent ange ledtrådar för att producera första hjärtat fält-liknande (FHF-L) stamfäder. Ännu viktigare, differentieras resulterande föräldraparets effektivt (~ 70 – 90%) till ventrikulära-liknande hjärtmuskelcellerna. Här beskriver vi en detaljerad metod för att 1) generera Id1-överuttryck hPSCs och 2) skilja skalbara mängder cryopreservable FHF-L stamfäder och ventrikulära-liknande hjärtmuskelcellerna.

Generation av hPSCs Id1 linjer hPSCs är infekterad med ett lentivirus som medla Id1 överuttryck (figur 1A). När hPSCId1 genereras, kvantifieras transgenens uttryck av qRT-PCR (figur 1B). Endast hPSCId1 rader uttrycka Id1 mRNA på nivåer som är större än 0,005 hudveck som för GAPDH bör...

Watch this Scientific Journal Video about Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells at JoVE.com

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

Här beskriver vi en skalbar metod, med en enkel kombination av Activin A och lentivirus-medierad Id1-överuttryck, för att generera första hjärtat fält-liknande hjärt stamfäder och ventrikulära-liknande hjärtmuskelcellerna från mänskliga pluripotenta stamceller.

Generation av första hjärtat fält-liknande hjärt stamfäder och ventrikulära-liknande hjärtmuskelcellerna från mänskliga pluripotenta stamceller

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

The main advantage of this simple technique is to generate cardiac progenitors and cardiomyocytes of defined heart field origin and subtypes. In turn, resulting cells can be used for a wide range of research application, including basic developmental biology, proof-of-concept cell-based therapy, and cardiac disease modeling studies. Demonstrating the procedure will be Sean Spiering and Michael Yu from my laboratory.To begin, coat enough wells of a 96-well plate with 50 microliters of coating reagent to accommodate subsequent cell seeding and viral infection steps, then add one milliliter of dissociation reagent to a well in the six-well plate to detach the pluripotent stem cells seeded. Then, use a sterile pipette to transfer the cell suspension in a 15-milliliter plastic centrifuge tube. In the tube, add five milliliters of PBS containing calcium and magnesium to neutralize the dissociation reagent.Then centrifuge the cells at 200 times g for three minutes. Post-centrifugation, discard the supernatant and then gently flick the tube for the pellet to be dislodged. Then dissolve the cell pellet in one milliliter of stem cell medium.Next, use an automated cell counter to count the number of cells. Once the counting is done, seed 20, 000 viable cells in each well of a 96-well plate containing 50 microliters of stem cell medium supplemented with two-micromolar RHO/ROCK pathway inhibitor. After 24 hours, monitor the cell attachment, then replace the medium with 100 microliters of stem cell medium one hour prior to the lentiviral infection.Next, thaw the Id1 lentivirus on ice, then add three microliters of purified lentivirus per well. After adding the lentivirus, transfer the plate back to cell culture incubator at 37 degrees Celsius with 5%carbon dioxide. 24 hours post-viral infection, add 100 microliters of fresh stem cell medium to the infected cells.After 48 hours, replace the virus-containing medium with 200 microliters of fresh stem cell medium supplemented with one microgram per milliliter of puromycin. Every day, keep changing the medium with fresh 200 microliters of stem cell medium supplemented with one microgram per milliliter of puromycin. Start the process of differentiation on Day 0 by replacing the stem cell medium with 1.5 milliliters of induction medium with Activin A in a 12-well plate.24 hours after differentiation has started, replace the medium with two milliliters of induction medium without Activin A.On the third day, replace with two milliliters of induction medium without Activin A.On the fifth day, aspirate the entire medium from the well containing the FHF-L CPs, then immediately add one milliliter of warm enzyme-containing dissociation reagent in the well, then incubate the culture plate at 37 degrees Celsius. After a minute, shake the plate inside the incubator and remove it from the incubator after two minutes, then add one milliliter of 10%fetal bovine serum-containing medium in the well. Detach the cells from the plate by gently pipetting the cells up and down.Next, transfer the cell suspension to a centrifuge tube and subject to centrifugation. After the centrifugation, dissolve the cell pellet in two milliliters of cryopreservation reagent, then count the cell number. Then transfer the cells in the cryopreservation vials and leave the vials in the cooling device.Then transfer the device at minus 80 degrees Celsius overnight. After 24 hours, transfer the vials in liquid nitrogen for long-term storage. Just before thawing the FHF-L CPs, coat the wells of the 12-well plate with two milliliters of coating reagent.Next transfer the five-days old FHF-L CPs from the liquid nitrogen in a dry ice container. From the dry ice, transfer the vial in a water bath at 37 degrees Celsius for two minutes to ensure complete thawing. After the cells are completely thawed, transfer the cells to a centrifuge tube, then pipette 10 drops of pre-warmed cardiogenic medium every 30 seconds to the cell solution.Keep adding the cardiogenic medium til the cryopreservation and cardiogenic medium ratio reaches one to three. Next subject the cells to centrifugation. Post-centrifugation remove the supernatant and carefully flick the tube, then dissolve the cell pellet in cardiogenic medium supplemented with two-micromolar RHO/ROCk pathway inhibitor.Further dilute the cell suspension with cardiogenic medium to achieve the desired cell concentration. Then seed the cells on the culture plate containing two milliliters of cardiogenic medium. Leave the plate in the tissue culture hood for 20 minutes before transferring to the incubator.Monitor the cell attachment on the sixth day of differentiation. On the seventh day, aspirate half of the cardiogenic medium and replace it with fresh medium. From here on out, replace 50%of the cardiogenic medium every other day, then, on the 15th day, quantify the cardiac differentiation.Here the expression level of Id1 mRNA is quantified in the hESC and hPSC cell lines. Only hPSC lines expressing Id1 mRNA at levels greater than 0.005-fold GAPDH are used for differentiation. Shown here are brightfield images post-differentiation of optimal, sub-confluent, and over-confluent hPSCs at Day 0.Upon further differentiation, the cell confluency reaches 75%after 24 hours. At Day 3, the differentiating cells form a cluster, and, by Day 5, the cells remain in tightly-connected clusters with homogenous morphology. At Day 6, the FHF-L CPs cover more than 90%of the surface area of the wells.In fact, by Day 12, the cardiomyocytes start to beat and display cardiac contractions. Further into the differentiation process, there are increased mRNA expression levels of MYL2 and IRX4 after Day 15. Both proteins represent ventricular-specific markers within the cardiomyocytes.Here the action potential trace represents the rapid upstroke and downstroke velocities as well as the ventricular plateau. These rates were characteristic of ventricular-like cardiomyocytes at Day 25 of differentiation. In summary, it takes about two weeks to generate Id1 over-expressing human pluripotent stem cells, then it takes about five days to generate first heart field-like progenitors and about 10 days to generate beating ventricular-like cardiomyocytes.

Research

JoVE Journal

Developmental Biology

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Härledning av renat Cardiomyocytes från Human inducerade pluripotenta stamceller Använda småmolekylära-module Differentiering och Efterföljande Glukos Svält

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Tredimensionell Kvantifiering av Dendritutskotten från Pyramidal nervceller Härstammar från Human inducerade pluripotenta stamceller

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Differentiering av Atrial Cardiomyocytes från pluripotenta stamceller med hjälp av BMP antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

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

Storskalig produktion av Cardiomyocytes från Human pluripotenta stamceller med hjälp av en mycket reproducerbar liten molekyl baserad differentiering protokoll

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

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Generering av inducerade pluripotenta stamceller från humant melanom tumörinfiltrerande lymfocyter

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Generation Integrations-fria inducerade pluripotenta stamceller från humant perifert blod mononukleära celler med användning av episomala vektorer

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Regisserad differentiering av primitiv och slutgiltig hematopoetiska progenitorceller från mänskliga pluripotenta stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Definierade och skalbar generation av hepatocyte-liknande celler från mänskliga pluripotenta stamceller

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

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

Differentierande kondrocyter från perifert blod härledda humana inducerade plöripotenta stamceller

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

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

Generation av standardiserade och reproducerbara framhjärnan-typ Cerebral Organoids från mänskliga inducerade pluripotenta stamceller

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...