Name: generation and expansion of human cardiomyocytes from patient peripheral blood mononuclear cells
Uploaded: 2021-02-12T15:00:00
Description: Watch this Scientific Journal Video about Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells at JoVE.com

Denne undersøgelse blev støttet af American Heart Association (AHA) Career Development Award 18CDA34110293 (M-T.Z.), Yderligere Ventures AVIF og SVRF awards (M-T.Z.), National Institutes of Health (NIH / NHLBI) giver 1R01HL124245, 1R01HL132520 og R01HL096962 (I.D.). Dr. Ming-Tao Zhao blev også støttet af start midler fra Abigail Wexner Research Institute på Nationwide Children's Hospital.

De eksperimentelle protokoller og informeret samtykke til mennesker blev godkendt af Institutional Review Board (IRB) på Nationwide Children's Hospital.
1. Forberedelse af cellekulturmedier, løsninger og reagenser
<ol><li>Forbered PBMC-medier<ol>
<li>Bland 20 mL basal PBMC kultur medier (1x) og 0,52 mL supplement. Der tilsættes hver 20 μL SCF og FLT3 (lagerkoncentration: 100 μg/mL), 4 μL IL3, IL6 og EPO (lagerkoncentration: 100 μg/mL) og 200 μL L-glutaminalternativ (100x). Bland dem ...

<ol>
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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=Titin+mutations+in+iPS+cells+define+sarcomere+insufficiency+as+a+cause+of+dilated+cardiomyopathy.">Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy.</a> Science. 349 (6251), 982-986 (2015).</li><li>Deacon, D. 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=Combinatorial+interactions+of+genetic+variants+in+human+cardiomyopathy.">Combinatorial interactions of genetic variants in human cardiomyopathy.</a> Nature Biomedical Engineering. 3 (2), 147-157 (2019).</li><li>Gifford, C. 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=Oligogenic+inheritance+of+a+human+heart+disease+involving+a+genetic+modifier.">Oligogenic inheritance of a human heart disease involving a genetic modifier.</a> Science. 364 (6443), 865-870 (2019).</li><li>Lo Sardo, V., 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=Unveiling+the+role+of+the+most+impactful+cardiovascular+risk+locus+through+haplotype+editing.">Unveiling the role of the most impactful cardiovascular risk locus through haplotype editing.</a> Cell. 175 (7), 1796-1810 (2018).</li><li>Liu, Y. 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=Human+embryonic+stem+cell-derived+cardiomyocytes+restore+function+in+infarcted+hearts+of+non-human+primates.">Human embryonic stem cell-derived cardiomyocytes restore function in infarcted hearts of non-human primates.</a> Nature Biotechnology. 36 (7), 597-605 (2018).</li><li>Zhao, M. T., Shao, N. Y., Garg, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-specific+cardiomyocytes+for+precision+medicine:+where+are+we+now.">Subtype-specific cardiomyocytes for precision medicine: where are we now.</a> Stem Cells. 38, 822-833 (2020).</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>Protze, S. I., Lee, J. H., Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pluripotent+stem+cell-derived+cardiovascular+cells:+from+developmental+biology+to+therapeutic+applications.">Human pluripotent stem cell-derived cardiovascular cells: from developmental biology to therapeutic applications.</a> Cell Stem Cell. 25 (3), 311-327 (2019).</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 (27), 1848-1857 (2012).</li><li>Zhao, M. 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=Molecular+and+functional+resemblance+of+differentiated+cells+derived+from+isogenic+human+iPSCs+and+SCNT-derived+ESCs.">Molecular and functional resemblance of differentiated cells derived from isogenic human iPSCs and SCNT-derived ESCs.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (52), 11111-11120 (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 (8), 855-860 (2014).</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=Chemically+defined,+albumin-free+human+cardiomyocyte+generation.">Chemically defined, albumin-free human cardiomyocyte generation.</a> Nature Methods. 12 (7), 595-596 (2015).</li><li>Buikema, J. 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=Wnt+activation+and+reduced+cell-cell+contact+synergistically+induce+massive+expansion+of+functional+human+ipsc-derived+cardiomyocytes.">Wnt activation and reduced cell-cell contact synergistically induce massive expansion of functional human ipsc-derived cardiomyocytes.</a> Cell Stem Cell. 27 (1), 50-63 (2020).</li><li>Lee, J. H., Protze, S. I., Laksman, Z., Backx, P. H., Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pluripotent+stem+cell-derived+atrial+and+ventricular+cardiomyocytes+develop+from+distinct+mesoderm+populations.">Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm populations.</a> Cell Stem Cell. 21 (2), 179-194 (2017).</li><li>Liang, 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=Canonical+Wnt+signaling+promotes+pacemaker+cell+specification+of+cardiac+mesodermal+cells+derived+from+mouse+and+human+embryonic+stem+cells.">Canonical Wnt signaling promotes pacemaker cell specification of cardiac mesodermal cells derived from mouse and human embryonic stem cells.</a> Stem Cells. 38 (3), 352-368 (2020).</li><li>Protze, S. 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=Sinoatrial+node+cardiomyocytes+derived+from+human+pluripotent+cells+function+as+a+biological+pacemaker.">Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker.</a> Nature Biotechnology. 35 (1), 56-68 (2017).</li><li>Ren, 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=Canonical+Wnt5b+signaling+directs+outlying+Nkx2.5++mesoderm+into+pacemaker+cardiomyocytes.">Canonical Wnt5b signaling directs outlying Nkx2.5+ mesoderm into pacemaker cardiomyocytes.</a> Developmental Cell. 50 (6), 729-743 (2019).</li><li>Zhang, 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=Direct+differentiation+of+atrial+and+ventricular+myocytes+from+human+embryonic+stem+cells+by+alternating+retinoid+signals.">Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals.</a> Cell Research. 21 (4), 579-587 (2011).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proceedings of the Japan Academy, Seriers B, Physical and Biological Sciences. 85 (8), 348-362 (2009).</li><li>Stacey, G. 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Under iPSC omprogrammering, er det afgørende for kultur PBMCs i 1 uge, indtil de er udvidet med klare kerner og cytoplasma. Da PBMCs ikke formerer sig, er et passende cellenummer til viral transduktion vigtigt for vellykket omprogrammering af iPSC. Celletal af PBMCs, mangfoldighed af infektion (MOI) og titer af virus bør overvejes og justeres for at nå de optimale transduktionsresultater. For hjertedifferentiering er den indledende såningstæthed afgørende for, at iPSCs når over 90% sammenløb den dag, hvor CHIR990...

Opdagelsen af menneskeskabte pluripotente stamceller har revolutioneret moderne hjerte-kar-medicin1,2. Humane iPSCs er i stand til selvfornyelse og generering af alle celletyper i hjertet, herunder kardiomyocytter, endotelceller, glatte muskelceller og hjertefibroblaster. Patient iPSC-afledte kardiomyocytter (iPSC-CMs) kan tjene som ubestemte ressourcer til modellering genetisk arvelige hjerte-kar-sygdomme (CVD'er) og test af hjertesikkerhed for nye lægemidler3. Især er patient iPSC-CMs godt klar til at undersøge genetiske og molekylære ætiologier af CVD'er, der er afledt af defekter i kardiomyocytter, såsom lange QT syndrom4 og udvidet kardiomyopati (DCM)5. Kombineret med CRISPR/Cas9-medieret genomredigering har patient iPSC-CMs åbnet en hidtil uset vej til at forstå det komplekse genetiske grundlag for CVD'er, herunder medfødte hjertefejl (CHDs)6,7,8. Human iPSC-CMs har også udstillet potentialer til at tjene som autologe celle kilder til genopfyldning af den beskadigede myokardie under et hjerteanfald9. I de senere år er det blevet altafgørende at generere højkvalitets menneskelige iPSC-CMs med definerede undertyper (atrie, ventrikulær og nodal) til hjerteregenerering og lægemiddeltest10.
Hjertedifferentiering fra menneskelige iPSCs har været meget avanceret i det seneste årti. Differentieringsmetoderne er gået fra embryoidkroppen (EB)-baseret spontan differentiering til kemisk defineret og styret hjertedifferentiering11. Centrale signalmolekyler, der er afgørende for embryonal hjerteudvikling, såsom Wnt, BMP, Nodal og FGF, manipuleres for at forbedre kardiomyocytdifferentiering fra humane iPSCs10,12. Væsentlige fremskridt omfatter sekventiel graduering af Wnt-signalering (aktivering efterfulgt af hæmning) for robust generering af kardiomyocytter fra humane iPSC'er13,14. Kemisk definerede hjertedifferentiering opskrifter er blevet undersøgt for at lette storstilet produktion af slå kardiomyocytter15,16, som har potentiale til at blive opgraderet til industriel og klinisk niveau produktion. Desuden opnås en robust udvidelse af tidlige humane iPSC-CMs ved at blive udsat for konstituerende Wnt-aktivering ved hjælp af et lille kemikalie (CHIR99021)17. Senest genereres subtypespecifikke kardiomyocytter gennem manipulation af retinsyre (RA) og Wnt-signalveje ved specifikke differentieringsvinduer under kardiomyocyt-afstamningforpligtelse fra humane iPSCs18,19,20,21,22.
I denne protokol beskriver vi en fungerende procedure for robust generering og spredning af menneskelige DM'er, der stammer fra patientens perifere blodmonnukleare celler. Vi præsenterer protokoller for 1) omprogrammering af menneskelige PBMCs til iPSCs, 2) robust generation af slå kardiomyocytter fra menneskelige iPSCs, 3) hurtig udvidelse af tidlige iPSC-CMs, 4) molekylær karakterisering af menneskelige iPSC-CMs, og 5) elektrofysiologisk måling af humane iPSC-CMs på enkeltcellet niveau ved patch clamp. Denne protokol dækker de detaljerede eksperimentelle procedurer for konvertering af patientens blodlegemer til at slå kardiomyocytter.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABI 7300 Fast Real-Time PCR System</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axon Axopatch 200B Microelectrode Amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Microelectrode Amplifier</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement minus insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm Fixation/Permeabilization Kit</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Fixation/Permeabilization solution, Perm/Wash buffer</td>
		</tr>
		<tr>
			<td>BD Vacutainer CPT tube</td>
			<td>BD Biosciences</td>
			<td>362753</td>
			<td>Blood cell separation tube</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Selleck Chemicals</td>
			<td>S2924</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTune-iPS 2.0 Sendai Reprogramming Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A16517</td>
			<td>Sendai virus reprogramming kit</td>
		</tr>
		<tr>
			<td>Digidata 1200B</td>
			<td>Axon Instruments</td>
			<td></td>
			<td>Acquisition board</td>
		</tr>
		<tr>
			<td>Direct-zol RNA Miniprep kit</td>
			<td>Zymo Research</td>
			<td>R2050</td>
			<td>RNA extraction kit</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1517001</td>
			<td>E8 media for iPSC culture</td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>L-glutamine alternative</td>
		</tr>
		<tr>
			<td>Growth factor reduced Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>iScript cDNA Snythesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708891</td>
			<td>cDNA synthesis</td>
		</tr>
		<tr>
			<td>IWR-1-endo</td>
			<td>Selleck Chemicals</td>
			<td>S7086</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement (KSR)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>pCLAMP 7.0</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Electrophysiology data acquisition &#38; analysis software</td>
		</tr>
		<tr>
			<td>Recombinant human EPO</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHC9631</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human FLT3</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHC9414</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL3</td>
			<td>Peprotech</td>
			<td>200-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL6</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHC0065</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human SCF</td>
			<td>Peprotech</td>
			<td>300-07</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, no glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>11879020</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFade Gold Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>S36936</td>
			<td>Mounting media</td>
		</tr>
		<tr>
			<td>StemPro-34 SFM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10639011</td>
			<td>PBMC culture media</td>
		</tr>
		<tr>
			<td>TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444964</td>
			<td>qPCR master mix</td>
		</tr>
		<tr>
			<td>TrypLE Select Enzyme 10x, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1217703</td>
			<td>CM dissociation solution</td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td>iPSC dissociation solution</td>
		</tr>
		<tr>
			<td>Y-27632 2HCl</td>
			<td>Selleck Chemicals</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and expansion of human cardiomyocytes from patient peripheral blood mononuclear cells

Generering patient-specifikke kardiomyocytter fra en enkelt blodprøve har tiltrukket enorm interesse i præcision medicin på hjerte-kar-sygdom. Hjertedifferentiering fra human inducerede pluripotente stamceller (iPSC'er) moduleres ved definerede signalveje, der er afgørende for embryonal hjerteudvikling. Talrige hjertedifferentieringsmetoder på 2D- og 3D-platforme er blevet udviklet med forskellige effektivitetsgevinster og kardiomyocytudbytte. Dette har undret efterforskere uden for feltet, da de mange forskellige af disse metoder kan være vanskelige at følge. Her præsenterer vi en omfattende protokol, der uddyber robust generering og udvidelse af patientspecifikke kardiomyocytter fra perifere blodmonnukleare celler (PBMCs). Vi beskriver først en højeffektiv iPSC-omprogrammeringsprotokol fra en patients blodprøve ved hjælp af sendai-virusvektorer uden integration. Vi beskriver derefter en lille molekylemedieret monolayer differentieringsmetode, der robust kan producere slå kardiomyocytter fra de fleste menneskelige iPSC-linjer. Derudover introduceres en skalerbar kardiomyocyt ekspansionsprotokol ved hjælp af et lille molekyle (CHIR99021), der hurtigt kan udvide patientafledte kardiomyocytter til industrielle og kliniske applikationer. I slutningen er detaljerede protokoller for molekylær identifikation og elektrofysiologisk karakterisering af disse iPSC-CMs afbildet. Vi forventer, at denne protokol er pragmatisk for begyndere med begrænset viden om hjerte-kar-udvikling og stamcellebiologi.

Human iPSC omprogrammering fra PBMCs Efter prækultur med Complete Blood Media i 7 dage bliver PBMCs store med synlige kerner og cytoplasma (Figur 1B), hvilket indikerer, at de er klar til virustransfektion. Efter transfection med Sendai virus omprogrammering faktorer, PBMCs vil gennemgå en epigenetisk omprogrammering proces for en anden uge. Typisk får vi 30-50 iPSC kolonier fra transfection af 1 x 105 PBMCs og omprogrammerin...

Watch this Scientific Journal Video about Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells at JoVE.com

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Her præsenterer vi en protokol til robust at generere og udvide menneskelige kardiomyocytter fra patientens perifere blodmonnukleare celler.

Generering og udvidelse af humane kardiomyocytter fra patientens perifere blod mononukleare celler

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Dette er omfattende protokol faciliteter, rå øger generation, og bruge pouncing af patient specifikke, kulturer fra perifere blod mononukleare celler. Denne taktik er reproducerbar og let at følge, specielt for begyndere. Og kræver ikke, specialekspertisere i stamcelle ballader, eller hjerte-kar-medicin.Vores protokol kan bruges til at generere patientspecifikke kardiomyocytter fra en enkelt blodprøve, der kan bruges til modellering af hjerte-kar-sygdomme og test af hjertetoksiciteten af nye lægemidler. Denne protokol kræver lang tid engagement i stamcelle omprogrammering vedligeholdelse og rettet differentiering. Men du sætter stor pris på dine bestræbelser.Når du ser slå kardiomyocytter i fadet. Når de menneskelige iPSC kolonier nå over 90% sammenløb, skylles hver brønd med tre milliliter DPBS før behandling af cellerne med en milliliter på 0,5 millimolar EDTA i DPBS per brønd ved 37 grader Celsius. Efter fem til otte minutter, tilføje en milliliter af iPSC passaging medium til hver brønd og manuelt løsne cellerne.Overfør 600 til 900 mikroliter af cellerne til individuelle Brønde har en kældermembran belagt seks brøndplade til en overnight inkubation ved 37 grader Celsius og 5% kuldioxid. Derefter opdatere kulturer med to milliliter af komplet E8 medium hver dag i tre til fire dage. Når kulturen har nået sammenløb, erstatte mediet med to milliliter af kardiomyocyt differentiering medium, to per brønd.På dag to, erstatte supernatants med to milliliter af kardiomyocyt differentiering medium en. På dag tre, erstatte supernatants med to milliliter af kardiomyocyt differentiering medium tre. På dag fem.Udskift supernatanten med to milliliter kardiomyocyt differentiering medium en. På dag syv til 10, erstatte supernatant med to milliliter af kardiomyocyt differentiering medium fire. På dag 11 og 13.Når der observeres kontraherende celler, skal supernatanten erstattes med to milliliter kardiomyocytdifferentiering mellem fem. På dag 15, 17, 19 og 21. Udskift supernatanten med to milliliter kardiomyocyt differentiering medium fire.Efter prækultur med komplet blodmedium pr. syv dage bliver PBMC stor med synlige kerner og cytoplasma, der indikerer, at de er klar til transfektion med Sendai Virus-omprogrammeringsfaktorer. Ved introduktion til komplet E8 medium. De helt omprogrammerede celler vil vedhæfte og begynde at danne kolonier.Efter fire til fem passager vil der dannes meget rene iPSC-kolonier fra de omprogrammerede celler med meget få observerede differentierede celler. På dette stadium, De fleste af cellerne er OCT4 og NANOG positive tegn på deres pluripoteni. Slå kardiomyocytter er normalt observeret efter 12 dages differentiering.Efter glukose sult, og replating iPSC kardiomyocytter udviser, spontane slå, og en justeret sarkom struktur med intercalated hjerte troponin T og alpha actinin udtryk. Kolonierne bevarer deres renhed, som det fremgår af deres Troponin T udtryk. Selv om iPSC kardiomyocytter er relativt umodne i forhold til voksne kardiomyocytter.De demonstrerer ventrikulære og atrielignende handlingspotentialer målt ved engros patch, klemme. Ventrikulære kardiomyocytter udtrykker myosin lyskæde to, mens atrie iPSC kardiomyocytter er præget af NR2F2 udtryk. Wnt aktivering stimulere celledeling og fremmer udtrykket af celle cyklus regulatorer.Interessant, Wnt aktivering også inducerer robust spredning af tidlige iPSC kardiomyocytter for to passager i forhold til kontrol, selv om denne proliferative evne mindskes over tid. Sørg for, at PBMS er udvidet med den klare nukleare og cytoplasma før iPSC omprogrammering vektorer er indført som betingelsen for PBMs er afgørende for iPSC omprogrammering. Patient specifikke kardiomyocytter er værdifulde for modellering hjerte-kar-sygdom in vitro og for at give et væld af donorceller til hjerte stamcelleterapi.

Research

JoVE Journal

Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Direkte Induktion af humane neurale stamceller fra perifert blod hæmatopoietiske stamceller

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Generering af induceret pluripotente stamceller fra humane perifere T-celler Brug af Sendai Virus i Feeder-fri Betingelser

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Isolation og udvidelse af mesenchymstamceller / stromacellers afledt af humane Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

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

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

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

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

Induceret Pluripotente Stem Cell Generation fra Blood Cells Brug Sendai Virus og Centrifugering

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

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

Generation af Integration-fri induceret pluripotente stamceller fra humane perifere mononucleære blodceller Brug episomale vektorer

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

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

Defineret og skalerbar Generation af hepatocyt-lignende celler fra menneskelige pluripotente 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

Differentierende chondrocytter fra perifert blodafledte humane inducerede pluripotente stamceller

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

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Isolation i Endothelial stamceller fra menneskelige navlestrengsblod

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

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

Generation af første hjertet felt-lignende hjerte ophav og ventrikel-lignende Cardiomyocytes fra humane pluripotente stamceller

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